Eleutherodactylus rayo a distinctive new species sharing features of the fitzingeri and rugulosus groups occurs along the Pacific slopes of the Cordillera de Talamanca of Costa Rica. Karyologically the new form most closely resembles El. vocalis of northwestern Mexico and certain populations of frogs from eastern Mexico, formerly referred to El. rugulosus but for which the name El. berkenbuschii W. Peters, 1870, is revived. Unlike other members of the ru- gulosus group which have 2N = 20, berkenbuschii, rayo and vocalis have 2N = 22 and approach El. talamancae of Coast Rica and Panama, a member of the fitzingeri group, in karyotypes.