The myiasis-causing flies Cuterebra grisea (Coquillet) and Cuterebra fontinella (Clark) (Diptera: Oestridae) are normally parasites of mice, predominantly of the genus Peromyscus. The morphological similarities of these species and the existence of intermediate morphotypes bearing characters of both species make the identification of adults problematic; furthermore the identification of larvae is apparently not possible. This study presents two molecular approaches to discriminate between these species using specific band patterns: (i) species-specific primers designed in the cytochrome oxidase II (COII) region used in multiplex polymerase chain reaction (PCR) and (ii) restriction fragment length polymorphism (RFLP) on amplified segments of cytochrome oxidase I (COI) gene. Both methods were tested on Cuterebra larvae and on adult museum specimens. The two techniques showed a clear difference between C. grisea and C. fontinella, although species-specific primers were more successful than RFLP for degraded DNA. No intraspecific variation in RFLP and species-specific amplifications were detected for the two species of Cuterebra. The results exhibit discrepancies between molecular and morphological identification, suggesting that some of the adults were misidentified.