The adult male and female of Polyplax guatemalensis sp. n. are described from the sigmodontine murid rodent Peromyscus grandis Goodwin collected in the Reserva de Biosfera, Sierra de las Minas, Guatemala, at an elevation of 2,200 m. The new species extends the number of known native species of Polyplax in the New World to four with none of them recorded south of Panama. Polyplax guatemalensis is morphologically most closely related to Polyplax auricularis which parasitises a cluster of closely related New World sigmodontine rodents from Canada to Panama. These two species can be distinguished from all other known species of Polyplax by the presence of partially overlapping, subtriangular, anterior abdominal plates in both sexes. Polyplax guatemalensis can be separated from P. auricularis by the abundant tergal abdominal setae and longer pseudopenis in males, and by the presence of one fewer anterior abdominal, subtriangular tergite and sternite in females.