diff --git a/gdb/remote.c b/gdb/remote.c
index 6d674c8139f..96e2750cd32 100644
--- a/gdb/remote.c
+++ b/gdb/remote.c
@@ -5636,6 +5636,15 @@ remote_unpush_target (remote_target *target)
       pop_all_targets_at_and_above (process_stratum);
       generic_mourn_inferior ();
     }
+
+  /* Don't rely on target_close doing this when the target is popped
+     from the last remote inferior above, because something may be
+     holding a reference to the target higher up on the stack, meaning
+     target_close won't be called yet.  We lost the connection to the
+     target, so clear these now, otherwise we may later throw
+     TARGET_CLOSE_ERROR while trying to tell the remote target to
+     close the file.  */
+  fileio_handles_invalidate_target (target);
 }
 
 static void
diff --git a/gdb/target.c b/gdb/target.c
index 767685fbca3..78752898191 100644
--- a/gdb/target.c
+++ b/gdb/target.c
@@ -3121,13 +3121,9 @@ static std::vector<fileio_fh_t> fileio_fhandles;
    list each time a new file is opened.  */
 static int lowest_closed_fd;
 
-/* Invalidate the target associated with open handles that were open
-   on target TARG, since we're about to close (and maybe destroy) the
-   target.  The handles remain open from the client's perspective, but
-   trying to do anything with them other than closing them will fail
-   with EIO.  */
+/* See target.h.  */
 
-static void
+void
 fileio_handles_invalidate_target (target_ops *targ)
 {
   for (fileio_fh_t &fh : fileio_fhandles)
diff --git a/gdb/target.h b/gdb/target.h
index e22f9038197..ddd4f3803cb 100644
--- a/gdb/target.h
+++ b/gdb/target.h
@@ -2230,6 +2230,12 @@ extern LONGEST target_fileio_read_alloc (struct inferior *inf,
 extern gdb::unique_xmalloc_ptr<char> target_fileio_read_stralloc
     (struct inferior *inf, const char *filename);
 
+/* Invalidate the target associated with open handles that were open
+   on target TARG, since we're about to close (and maybe destroy) the
+   target.  The handles remain open from the client's perspective, but
+   trying to do anything with them other than closing them will fail
+   with EIO.  */
+extern void fileio_handles_invalidate_target (target_ops *targ);
 
 /* Tracepoint-related operations.  */
 
diff --git a/gdb/testsuite/gdb.base/detach-sysroot-target.c b/gdb/testsuite/gdb.base/detach-sysroot-target.c
new file mode 100644
index 00000000000..9811b15f06d
--- /dev/null
+++ b/gdb/testsuite/gdb.base/detach-sysroot-target.c
@@ -0,0 +1,22 @@
+/* This testcase is part of GDB, the GNU debugger.
+
+   Copyright 2021 Free Software Foundation, Inc.
+
+   This program is free software; you can redistribute it and/or modify
+   it under the terms of the GNU General Public License as published by
+   the Free Software Foundation; either version 3 of the License, or
+   (at your option) any later version.
+
+   This program is distributed in the hope that it will be useful,
+   but WITHOUT ANY WARRANTY; without even the implied warranty of
+   MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.  See the
+   GNU General Public License for more details.
+
+   You should have received a copy of the GNU General Public License
+   along with this program.  If not, see <http://www.gnu.org/licenses/>.  */
+
+int
+main ()
+{
+  return 0;
+}
diff --git a/gdb/testsuite/gdb.base/detach-sysroot-target.exp b/gdb/testsuite/gdb.base/detach-sysroot-target.exp
new file mode 100644
index 00000000000..9ae1eb6277a
--- /dev/null
+++ b/gdb/testsuite/gdb.base/detach-sysroot-target.exp
@@ -0,0 +1,59 @@
+# Copyright 2021 Free Software Foundation, Inc.
+
+# This program is free software; you can redistribute it and/or modify
+# it under the terms of the GNU General Public License as published by
+# the Free Software Foundation; either version 3 of the License, or
+# (at your option) any later version.
+#
+# This program is distributed in the hope that it will be useful,
+# but WITHOUT ANY WARRANTY; without even the implied warranty of
+# MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.  See the
+# GNU General Public License for more details.
+#
+# You should have received a copy of the GNU General Public License
+# along with this program.  If not, see <http://www.gnu.org/licenses/>.  */
+
+# Test running a program from the GDB prompt and then detaching it,
+# with "set sysroot target:".  Regression test for PR gdb/28080.
+#
+# It is assumed that it is only safe to tweak the sysroot on the
+# current board if it is currently either empty or already "target:".
+# If set to anything else, we skip the test.
+
+standard_testfile
+
+if {[prepare_for_testing "failed to prepare" ${binfile} ${srcfile}]} {
+    return
+}
+
+gdb_test_multiple "show sysroot" "" {
+    -wrap -re "The current system root is \"\"\\." {
+	pass $gdb_test_name
+
+	# Explicitly set target: sysroot.
+	gdb_test_no_output "set sysroot target:"
+    }
+    -wrap -re "The current system root is \"target:\"\\." {
+	pass $gdb_test_name
+
+	# Nothing else to do.
+    }
+    -re "$gdb_prompt $" {
+	pass $gdb_test_name
+
+	# If testing with any other sysroot, we probably should not
+	# mess with it.
+	unsupported "sysroot is set"
+	return
+    }
+}
+
+if ![runto_main] {
+    fail "couldn't run to main"
+    return
+}
+
+# With PR gdb/28080, this would crash GDB when testing with "target
+# remote".
+set escapedbinfile [string_to_regexp ${binfile}]
+gdb_test "detach" "Detaching from program: .*$escapedbinfile, .*"