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The largemouth bass (Micropterus salmoides) has become an important freshwater fish in China since it was intentionally introduced in the 1980 s, and the amount produced in aquaculture is increasing each year. Females are preferred because they grow faster than males and reach a larger body size. However, the physiological sex cannot be accurately determined from external morphology at the early growth stage, so it is crucial to develop sex-specific molecular markers. In this study, a single nucleotide polymorphism (SNP) marker was developed based on whole genome resequencing data obtained from 30 females and 30 males. This marker made it possible to positively identify male and female largemouth bass. Through Fst value screening, 123 SNP loci significantly associated with sex were obtained, and nearly 40% of them were located on chromosome 7 in a 3.44-Mb sex-linked region. A method of polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) combined with a TaqMan minor groove binder (MGB) probe was developed based on a sex-specific marker. Using this method, male and female fish in four different populations (n = 128) were identified with 100% accuracy. Compared with PCR-RFLP followed by electrophoretic separation of fragments, the PCR-RFLP–TaqMan MGB probe method reduced the time required to identify the sex of largemouth bass. The validation results also indicated that largemouth bass has an XX/XY sex-determination system. In conclusion, we have identified a sex-specific SNP marker for largemouth bass and developed a new technique to identify the sex of largemouth bass rapidly and accurately. These findings provide a scientific basis for further research on mono-sex breeding and the molecular mechanism of sex determination in largemouth bass.