ABSTRACT Agromyza Fallén (Diptera, Agromyzidae) is a genus of leaf mining flies, including species with high economic importance. The knowledge of this genus is very poor in the neotropics, with 12 known species, only six of them recorded from Brazil. This paper describes two new Agromyza species from "Cerrado" and "Pantanal" biomes and records three other species represented only by females that could not be identified to species level. We also present a taxonomic key to segregate the 14 Neotropical species. The specimens were collected in Mato Grosso and Mato Grosso do Sul states and are deposited at Museu de Zoologia, Universidade de São Paulo (São Paulo, Brazil) and Museu Nacional, Universidade Federal do Rio de Janeiro (Rio de Janeiro, Brazil) collections. The adults were photographed and the male terminalia were dissected and illustrated.