Biological characteristics of two entities of the Anastrepha fraterculus complex (Diptera: Tephritidae), referred to in previous publications as A. sp.1 aff. fraterculus and A. sp.2 aff. fraterculus, were further studied by a combined analysis of isozymes, karyotypes, morphometry, and crossings, in samples from 10 Brazilian populations. A survey of 16 enzymatic systems comprising 19 loci showed significant differences in the allele frequencies at four loci, FUM, ME, HEX, and LDH, allowing the recognition of two population clusters. These clusters also differ in their karyotypes, especially in the length of the sex chromosomes and in the size and location of heterochromatic regions. A morphometric analysis of wings and the aculeus in samples from five populations clearly showed a distinction between the two clusters but not between populations within each cluster. A phenetic analysis based on the Mahalanobis distance matrix also arranged the populations into the same two clusters. Crosses between populations of the same cluster showed no significant differences in egg hatching and in the adult sex ratio. However, a significant decrease in egg hatching was observed in the intercluster crosses. In crosses of cluster 1 males to cluster 2 females, a significant deviation in the sex ratio was observed according to Haldane’s rule, but not in the reciprocal crosses, indicating that a certain degree of reproductive isolation occurs between populations of cluster 1 and cluster 2. The results indicate that the two population clusters actually represent two cryptic species of the nominal species Anastrepha fraterculus, corroborating previous studies on this complex. We propose that the denominations A. sp.1 aff. fraterculus and A. sp.2 aff. fraterculus should be maintained until an appropriate taxonomic revision is made.