The apple maggot, Rhagoletis pomonella (Walsh), was introduced into the apple-growing regions of the Pacific Northwest in the U.S.A. during the past 60–100 yr. Apple maggot (larvae, puparia, and adults) is difficult to distinguish from its morphologically similar sister species, Rhagoletis zephyria Snow, which is native and abundant in the Pacific Northwest. While morphological identifications are common practice, a simple, inexpensive assay based on genetic differences would be very useful when morphological traits are unclear. Here we report nucleotide substitution and insertion–deletion mutations in the nontranscribed spacer (NTS) of the ribosomal RNA gene cistron of R. pomonella and R. zephyria that appear to be diagnostic for these two fly species. Insertion–deletion variation is substantial and results in a 49 base-pair difference in PCR amplicon size between R. zephyria and R. pomonella that can be scored using agarose gel electrophoresis. PCR amplification and DNA sequencing of 766 bp of the NTS region from 38 R. pomonella individuals and 35 R. zephyria individuals from across their geographic ranges led to the expected PCR fragments of approx. 840 bp and 790 bp, respectively, as did amplification and sequencing of a smaller set of 26 R. pomonella and 16 R. zephyria flies from a sympatric site in Washington State. Conversely, 633 bp mitochondrial COI barcode sequences from this set of flies were polyphyletic with respect to R. pomonella and R. zephyria. Thus, differences in NTS PCR products on agarose gels potentially provide a simple way to distinguish between R. pomonella and R. zephyria.