Phymatotrichopsis omnivora is a member of Pezizomycetes and causes root rot disease on a broad range of dicotyledonous plants. Using recently generated draft genome sequence data from four P. omnivora isolates, we developed simple sequence repeat (SSR) markers and identified both mating type genes (MAT1-1-1 and MAT1-2-1) in this fungus. To understand the genetic diversity of P. omnivora isolates (n = 43) and spore mats (n = 29) collected from four locations (Oklahoma, Texas, Arizona, and Mexico) and four host crops (cotton, alfalfa, peach, and soybean), we applied 24 SSR markers and showed that of the 72 P. omnivora isolates and spore mats tested, 41 were distinct genotypes. Furthermore, the developed SSR markers did not show cross-transferability to other close relatives of P. omnivora in the class Pezizomycetes. A multiplex PCR detecting both mating type idiomorphs and a reference gene (TUB2) was developed to screen P. omnivora isolates. Based on the dataset we tested, P. omnivora is a heterothallic fungus with both mating types present in the United States in a ratio close to 1:1. We tested P. omnivora spore mats obtained from spatially distinct disease rings that developed in a center-pivot alfalfa field and showed that both mating types can be present not only in the same field but also within a single spore mat. This study shows that P. omnivora has the genetic toolkit for generating sexually diverse progeny, providing impetus for future studies that focus on identifying sexual morphs in nature.