diff --git a/CHANGES.md b/CHANGES.md
index 44159bbbe..3ce503a09 100644
--- a/CHANGES.md
+++ b/CHANGES.md
@@ -3,6 +3,9 @@
 ## __NEXT__
 
 * ancestral: For VCF alignments, a VCF output file is now only created when requested via `--output-vcf`. [#1344][] (@jameshadfield)
+* ancestral: Improvements to command line arguments. [#1344][] (@jameshadfield)
+     * Incompatible arguments are now checked, especially related to VCF vs FASTA inputs. 
+     * `--vcf-reference` and `--root-sequence` are now mutually exclusive.
 
 [#1344]: https://github.com/nextstrain/augur/issues/1334
 
diff --git a/augur/ancestral.py b/augur/ancestral.py
index 260c252d6..d2012342c 100644
--- a/augur/ancestral.py
+++ b/augur/ancestral.py
@@ -180,9 +180,13 @@ def register_parser(parent_subparsers):
     )
     input_group.add_argument('--tree', '-t', required=True, help="prebuilt Newick")
     input_group.add_argument('--alignment', '-a', help="alignment in FASTA or VCF format")
-    input_group.add_argument('--vcf-reference', type=str, help='FASTA file of the sequence the VCF was mapped to (only used if a VCF is provided as the alignment)')
-    input_group.add_argument('--root-sequence', type=str, help='FASTA/genbank file of the sequence that is used as root for mutation calling.'
-                        ' Differences between this sequence and the inferred root will be reported as mutations on the root branch.')
+    input_group_ref = input_group.add_mutually_exclusive_group()
+    input_group_ref.add_argument('--vcf-reference', type=str, metavar='FASTA',
+                                 help='[VCF alignment only] file of the sequence the VCF was mapped to.'
+                                      ' Differences between this sequence and the inferred root will be reported as mutations on the root branch.')
+    input_group_ref.add_argument('--root-sequence', type=str,metavar='FASTA/GenBank',
+                                 help='[FASTA alignment only] file of the sequence that is used as root for mutation calling.'
+                                      ' Differences between this sequence and the inferred root will be reported as mutations on the root branch.')
 
     global_options_group = parser.add_argument_group(
         "global options",
diff --git a/tests/functional/ancestral/cram/invalid-args.t b/tests/functional/ancestral/cram/invalid-args.t
index e91132835..888b66672 100644
--- a/tests/functional/ancestral/cram/invalid-args.t
+++ b/tests/functional/ancestral/cram/invalid-args.t
@@ -51,3 +51,18 @@ Missing tree file
   >  --output-sequences "$CRAMTMP/$TESTFILE/output.fasta" > /dev/null
   ERROR: The provided tree file .* doesn't exist (re)
   [2]
+
+
+Attempting to use FASTA-input reference and VCF-input reference args
+(The files here don't exist, but we exit before they're checked) 
+
+  $ ${AUGUR} ancestral \
+  >  --tree $TESTDIR/../data/tree-doesnt-exist.nwk \
+  >  --alignment $TESTDIR/../data/aligned.fasta \
+  >  --root-sequence $TESTDIR/../data/reference.fasta \
+  >  --vcf-reference $TESTDIR/../data/reference.fasta \
+  >  --output-sequences "$CRAMTMP/$TESTFILE/output.fasta" > /dev/null 2>"$CRAMTMP/$TESTFILE/err-args.txt"
+  [2]
+
+  $ grep "augur ancestral: error: argument --vcf-reference: not allowed with argument --root-sequence" "$CRAMTMP/$TESTFILE/err-args.txt"
+  augur ancestral: error: argument --vcf-reference: not allowed with argument --root-sequence