Molecular methods are necessary to diagnose immature life stages of the agricultural pest fruit fly Bactrocera dorsalis (Hendel), and are useful to corroborate identifications based on adults because morphological variation within the species can overlap with congeners. DNA sequencing of the nuclear ribosomal internal transcribed spacer 1 (ITS-1) has been adopted by the International Plant Protection Convention as an internationally accepted method to distinguish between the 2 pestiferous fruit fly species Bactrocera dorsalis and Bactrocera carambolae (Drew & Hancock). Reported ITS-1 sequences also are distinct and diagnostically informative to distinguish several other Bactrocera species related to B. dorsalis. In this study, we applied DNA sequencing of ITS-1 to a collection of 513 adult flies trapped in California, USA, in the yr 2008 to 2018. Internal transcribed spacer 1 sequences were successfully recovered from 504 (98%) of these flies. One fly had an ITS-1 sequence that matched B. occipitalis (Bezzi) records. Re-examination of that fly using cytochrome c oxidase I, elongation factor 1-alpha, and morphology supports it as the second record of B. occipitalis trapped in California. The other 503 flies had ITS-1 sequences consistent with B. dorsalis. Six unique ITS-1 sequences (or DNA types) were observed in the collection of 503 B. dorsalis. Three of the ITS-1 sequences (types A, B, and C) were present in 84% of the 503 flies and match ITS-1 records reported in prior publications on B. dorsalis. The other 3 sequences (types D, E, and F) observed in 4% of the 503 B. dorsalis have not been reported in publications. Ambiguous nucleotides were observed from 12% of the 503 B. dorsalis flies, precluding designation of a sequence type. Including the 3 new types from the current study, a total of 15 unique ITS-1 sequences now are known for B. dorsalis. The study, therefore, documents additional intraspecific variation of ITS-1 that aids in future applications for species identification.