The taxonomic status of two closely related species of Latrodectus, L. mactans and L. hesperus, has been debated for many years. Based on morphological characteristics and genitalia, some workers consider them as valid species and others as subspecies. This study was conducted to determine whether the internal transcribed spacers 2 (ITS2) of rDNA exhibit sequence differences between the two taxa that could delineate their taxonomic relationship. Individuals of L. mactans and L. hesperus from six populations were collected and identified based on morphological characteristics. The ITS2 rDNA of nine individuals was sequenced and analyzed. Results indicate that the minimal differences present in the ITS2 sequences are taxonomically insignificant.