Elassoma gilberti is distinguished from its close relative E. okefenokee by possessing four pores in the preopercular (PO) canal on each side of the head and usually seven anal fin rays. Elassoma okefenokee has three PO pores and usually eight anal fin rays. Elassoma gilberti has slightly less deep body and slightly smaller dorsal and anal fins than E. okefenokee. Breeding females of E. gilberti often have blue dashes below and behind the eye, which are lacking in female E. okefenokee. Otherwise, the two species are almost identical or broadly overlapping in meristic, morphometric, and color features. The distinction of the two species is supported by molecular data. Eight fixed differences (of 553 bp assayed) at the mitochondrial 16S rRNA locus and 12 fixed differences (of 583 bp assayed) found at the nuclear S7 locus differentiated the two species. Phylogenetic analyses using these molecular characters supported monophyletic clades that contained haplotypes and alleles found uniquely in E. gilberti and E. okefenokee, respectively.