It is firmly believed that ancestral primates were nocturnal, with nocturnality having been maintained in most prosimian lineages. Under this traditional view, the opsin genes in all nocturnal prosimians should have undergone similar degrees of functional relaxation and accumulated similar extents of deleterious mutations. This expectation is rejected by the short-wavelength (S) opsin gene sequences from 14 representative prosimians. We found severe defects of the S opsin gene only in lorisiforms, but no defect in five nocturnal and two diurnal lemur species and only minor defects in two tarsiers and two nocturnal lemurs. Further, the nonsynonymous-to-synonymous rate ratio of the S opsin gene is highest in the lorisiforms and varies among the other prosimian branches, indicating different time periods of functional relaxation among lineages. These observations suggest that the ancestral primates were diurnal or cathemeral and that nocturnality has evolved several times in the prosimians, first in the lorisiforms but much later in other lineages. This view is further supported by the distribution pattern of the middle-wavelength (M) and long-wavelength (L) opsin genes among prosimians.