The Gambusia yucatana complex (sensu Greenfield and Wildrick, 1984) was reviewed and shown to consist of two subspecies. Principal component analysis demonstrated that the populations from the northern Yucatan Peninsula form a group that is distinct from other populations directly to the south. The northern Yucatan Peninsula populations represent the nominate subspecies G. yucatana yucatana. The populations from the lake region of the Peten District of Guatemala, although occurring far to the south of the Yucatan Peninsula, cluster with the populations of G. yucatana yucatana and thus are included in that subspecies pending further investigation. The populations from the southern Yucatan Peninsula, southern Mexico and Belize are recognized as G. yucatana australis n. subsp. Laboratory rearing has demonstrated that the characters utilized to differentiate the subspecies are not the result of environmental induction of phenotypes. The status of the deepand slender-bodied forms of the G. yucatana subspecies is discussed. Although these differences have been shown to have a genetic basis, it is argued that slender-bodied or deep-bodied forms should not be given taxonomic rank on the basis of that character alone.