Recent collections from the Conasauga River system revealed the presence of Gambusia holbrooki, previously unreported in the Coosa River portion of the Mobile River drainage. We analyzed Gambusia specimens in 36 collections from the Conasauga River system to determine the relative distribution of G. affinis, G. holbrooki, or their hybrid progeny. We used gonopodial characters and ray counts from the anal and dorsal fins, which required reconciling differences among published accounts, to make species determinations. We counted all fin rays separately for dorsal and anal fins and recommend this method to clarify discrepancies among previous reports. This method yielded counts of seven dorsal and 10 anal rays for G. affinis and eight dorsal and 11 anal rays for G. holbrooki. Both species and their hybrids were collected in the Conasauga River system. Gambusia affinis is widespread in the Conasauga River system and is presumably native. Gambusia holbrooki currently has a limited distribution but appears to be expanding its range. We attribute the occurrence of G. holbrooki to human introduction.