Drosophila butantan sp. nov., a species belonging to the guarani group and closely related to Drosophila igrifemur from Bolivia, is described based on a female, and some of its offspring, collected at the forest reserve of the Instituto de Biociências da Universidade de São Paulo, Cidade Universitária “Armando de Salles Oliveira”, São Paulo City, state of São Paulo, Brazil. Although externally similar, the two apparently forest-dwelling species can be told apart by having distinct oviscapt valves and spermathecal introverts and tips. Accordingly, a proposal is made to also include D. nigrifemur, a previously unassigned species, in the guarani group. The two species seem to be also related to Drosophila alexandrei and Drosophila guaraja as indicated by their external morphology, their elongate spermathecae and the not so sharply pointed oviscapt valves. The karyotypes of the new species differ from those described for D. alexandrei and D. guaraja, while those of D. nigrifemur remain still unknown. Photomicrographs of the male and female imagines, in addition to drawings and photos of their terminalia, are also included.