G. holbrooki usually has seven dorsal rays and a gonopodium with prominent teeth on ray three. G. affinis usually has six dorsal rays and lacks prominent teeth on gonopodial ray three. Both subspecies have a chromosome number of 2n = 48 but female G. affinis possess a large heteromorphic sex chromosome which is lacking in G. holbrooki (Black and Howell 1979).
In Arizona, G. affinis may be confused with Poeciliopsis occidentalis, the Sonoran topminnow. In topminnows the gonopodium is asymmetrical to the left, large hooks and serrae absent on gonopodial tip, and the gonopodium reaches beyond the snout when directed forward. The pelvic fins of males are unmodified and somewhat reduced. Many breeding males will be blackened. In G. affinis the gonopodium is symmetrical with large hooks and serrae on the tip. The pelvic fins of males are modified with a fleshy appendage on the distal third of the first, short, unbranched ray. Males are rarely blackened.