diff --git a/python/CHANGELOG.rst b/python/CHANGELOG.rst
index a6f5986e62..2fb1eb9459 100644
--- a/python/CHANGELOG.rst
+++ b/python/CHANGELOG.rst
@@ -19,6 +19,11 @@
   and a window breakpoint falls within an internal missing interval.
   (:user:`nspope`, :pr:`3176`, :issue:`3175`)
 
+**Breaking changes** 
+
+- ``ltrim``, ``rtrim``, ``trim`` and ``shift`` raise an error if used on a tree sequence
+  containing a reference sequence (:user:`hyanwong`, :pr:`3210`, :issue:`2091`)
+
 --------------------
 [0.6.4] - 2025-05-21
 --------------------
diff --git a/python/tests/test_topology.py b/python/tests/test_topology.py
index b2eb4ea6a7..89d72d31fe 100644
--- a/python/tests/test_topology.py
+++ b/python/tests/test_topology.py
@@ -7074,6 +7074,18 @@ def test_failure_with_migrations(self):
         with pytest.raises(ValueError):
             ts.trim()
 
+    def test_reference_sequence(self):
+        # Test that we fail if there is a reference sequence
+        tables = tskit.TableCollection(3.0)
+        tables.reference_sequence.data = "ABC"
+        ts = tables.tree_sequence()
+        with pytest.raises(ValueError, match="reference sequence"):
+            ts.ltrim()
+        with pytest.raises(ValueError, match="reference sequence"):
+            ts.rtrim()
+        with pytest.raises(ValueError, match="reference sequence"):
+            ts.trim()
+
 
 class TestShift:
     """
@@ -7140,6 +7152,14 @@ def test_bad_seq_len(self):
         ):
             ts.shift(1, sequence_length=1)
 
+    def test_reference_sequence(self):
+        # Test that we fail if there is a reference sequence
+        tables = tskit.TableCollection(3.0)
+        tables.reference_sequence.data = "ABC"
+        ts = tables.tree_sequence()
+        with pytest.raises(ValueError, match="reference sequence"):
+            ts.shift(1)
+
 
 class TestConcatenate:
     def test_simple(self):
diff --git a/python/tskit/tables.py b/python/tskit/tables.py
index bc078164c0..0ba33061fe 100644
--- a/python/tskit/tables.py
+++ b/python/tskit/tables.py
@@ -3887,6 +3887,11 @@ def keep_intervals(self, intervals, simplify=True, record_provenance=True):
             )
 
     def _check_trim_conditions(self):
+        if self.has_reference_sequence():
+            raise ValueError(
+                "Cannot trim if there is a reference sequence. Please remove the "
+                "reference sequence by calling `.reference_sequence.clear()` first."
+            )
         if self.migrations.num_rows > 0:
             if (np.min(self.migrations.left) < np.min(self.edges.left)) and (
                 np.max(self.migrations.right) > np.max(self.edges.right)
@@ -4005,6 +4010,11 @@ def shift(self, value, *, sequence_length=None, record_provenance=True):
         :param sequence_length: The new sequence length of the tree sequence. If
             ``None`` (default) add `value` to the sequence length.
         """
+        if self.has_reference_sequence():
+            raise ValueError(
+                "Cannot shift if there is a reference sequence. Please remove the "
+                "reference sequence by calling `.reference_sequence.clear()` first."
+            )
         self.drop_index()
         self.edges.left += value
         self.edges.right += value