The Middle American killifish genus Profundulus occurs in most Pacific and Atlantic drainages from the Mexican state of Guerrero to the Chamelecón River in Honduras, with highest species diversity in southern Mexico. In this study, we describe a new member of the genus, Profundulus parentiae, new species, from the Mexican state of Oaxaca. It is distinguished from P. guatemalensis and P. kreiseri by having rows of dots on the sides of the body. Profundulus parentiae, new species, can be distinguished from P. oaxacae by having 33 lateral scales, versus 29–31 in P. oaxacae. Profundulus parentiae, new species, can be distinguished from P. balsanus, P. oaxacae, and P. mixtlanensis by the presence of long epiotic processes that extend beyond the epipleural ribs of the first vertebra, versus short epiotic processes not reaching the epipleural ribs of the first vertebra. Profundulus parentiae, new species, can be differentiated from P. punctatus by the presence of a dorso-ventrally compressed Meckel's cartilage with a relatively straight medial process and a narrow and strongly concave sesamoid articular, versus a dorso-ventrally expanded Meckel's cartilage with concave medial process and a wide and moderately concave sesamoid articular. Based on the phylogenetic analysis of molecular sequence data, the new species is recovered as the sister taxon to P. balsanus. The discovery and description of this new species in southeastern Mexico, raises the number of species from this region to five and suggests that this area has been an important center for diversification within this lineage of killifishes.