Analysis of mosquitofish, Gambusia affinis, has proven useful for monitoring contaminant levels in aquatic biota; however, the small size of this species often requires the compositing of several fish to provide sufficient biomass for selenium analysis. Such composites have usually been obtained without considering the length and sex of the individual fish. The present study found significant differences in mean lengths and sex ratios of mosquitofish sampled with small-mesh dip nets from five sites close to each other in the San Joaquin Valley, California. To test the effect of these different sample characteristics, fish from each site were divided by sex into five size classes (< 20, 20–30, 31–45, 46–60, and > 60 mm total length) before analysis for total selenium. Altogether, fish from the San Luis Drain and Kesterson Ponds 2 and 7 contained 65–360 μg g −1 selenium (dry weight basis), or about 28- to 300-fold more than concentrations in fish of the same length and sex from the Volta Wasteway and Volta Pond 26. Except for females 31–45 mm long from the San Luis Drain that had higher concentrations than either smaller or larger females, selenium concentrations did not differ significantly among size classes. Although concentrations differed between sexes in samples from the San Luis Drain, Kesterson Pond 2, and Volta Pond 26, neither males nor females consistently had the higher concentrations. In the San Luis Drain, 20–30 and 31–45 mm long females had higher concentrations than did males of the same size classes; in Kesterson Pond 2 and Volta Pond 26, however, 20–30 mm long males had higher concentrations than did females of the same size class. Although no consistent patterns were observed, the occasional differences in selenium concentrations in fish of different length and sex indicate that these variables should be considered when surveys and monitoring studies are designed.