In this study, we provide the first report of an unisexual population of the lizard genus Cnemidophorus south of the Amazon forest and the first in South America not belonging to the C. lemniscatus complex. The population was found in a relatively small open sandy area (approximately 1.8 km in diameter) covered with herbaceous vegetation inside the Atlantic forest in southeastern Brazil. Collections made in four different years (1991, 1993, 1994, and 1995) contained only females (n = 42). Cytogenetic analysis showed a karyotype of 2N = 48 (five submetacentric; 19 telocentric and subtelocentric macrochromosomes; 24 microchromosomes). There are structural heterozygotes in the second, seventh, and thirteenth pairs of chromosomes. The absence of males in the samples and the karyotype pattern indicate that this population is another unisexual species of Cnemidophorus and the first case in South America belonging to the Cnemidophorus ocellifer complex. The new entity is described as a new species.