Macrophomina phaseolina is responsible for significant yield loss in soybean worldwide and is capable of infecting more than 500 plant species including maize. Despite its broad host range, M. phaseolina reproduces exclusively through asexual means. The objectives of this study were to phenotype and genotype populations of M. phaseolina associated with soybean in order to understand the genetic basis of adaptation in this asexual pathogen. A total of 200 M. phaseolina isolates were obtained from soil collected from soybean and corn fields in Ohio and 300 isolates from soybean fields in Paraguay. We assessed pathogenicity on soybean and in vitro growth at various temperatures, and then sequenced the genomes of 12 selected isolates using a combination of short and long read technologies to obtain near-chromosome level assemblies. By building SNP-based phylogenies derived from whole genome alignments, we identified two major populations: one containing only isolates from Ohio and another containing isolates from both Ohio and Paraguay. Although all 12 isolates were nearly identical (>97%) at the nucleotide level, we found that genome size differed by as much as 25%, and predicted gene number varied by 30%. We also found that M. phaseolina’s pangenome is more open than the pangenomes of other obligate asexual fungi, suggesting that this pathogen has retained mechanisms for generating genotypic diversity despite its obligate asexuality.