The species of the South American genus Souzalopesmyia Albuquerque, 1951 (Diptera: Muscidae) are very similar externally, but can be distinguished by characters of the terminalia of the males. We describe Souzalopesmyia polleti n. sp. from French Guiana and provide diagnostic images for it. The new species can be distinguished from all other species of the genus by the following combination of characters: male with two long pairs of frontal setae; one weak proepisternal seta, about ¾ length of upper anepisternal setae; upper postpronotal setae longer than half length of lower one; cercal plate with margins of the posterior incision divergent; sternite 5 with posterior depression and phallapodeme enlarged on distal region. An updated phylogenetic analysis based on morphological evidence, yielded a single most parsimonious tree (S. amazonica Albuquerque, 1951 + (S. carioca Albuquerque, 1951 + (S. paraensis de Carvalho, 1999 + S. polleti n. sp.) + (S. singularis (Stein, 1919) + S. sulina de Carvalho, 1999))). A new record of Souzalopesmyia paraensis from Peru, maps of the species’ distribution, an updated distribution of S. singularis and an updated identification key are also presented.