Profundulus adani, new species, is described from the upper reaches of the Papaloapan River in the Mexican state of Oaxaca. The uniqueness of this new species is supported by morphological and molecular evidence. A combination of color patterns and counts separate P. adani, new species, from its congeners. Profundulus adani, new species, is distinguished from all congeners by the absence of a humeral spot in individuals larger than 45 mm SL. It can further be distinguished from P. balsanus, P. chimalapensis, P. kreiseri, P. oaxacae, P. parentiae, and P. punctatus based on the absence of series of dark dots on the sides of the body. Profundulus adani, new species, shows a faded dark band that extends from the tip of the operculum to the end of the caudal peduncle; this band is absent in P. balsanus and P. parentiae. The new species is described using specimens collected in the upper reaches of the Papaloapan River in the Mexican state of Oaxaca; geographically this represents a significant range expansion and extends the Atlantic slope northernmost limit of the Profundulidae.