Over half of U.S. cucurbits are produced in the southeastern states; however, crops in this region are exposed to a destructive foliar disease called gummy stem blight (GSB). A recent study revealed that GSB is caused by three morphologically similar, but genetically distinct Stagonosporopsis species: S. citrulli, S. cucurbitacearum, and S. caricae. In order to understand the spatial genetic structure of GSB fungi in the southeastern U.S., 528 isolates collected from nine fields over three years were genotyped with 16 microsatellite markers. S. caricae was reported for the first time in the southeastern U.S. Populations of the dominant species S. citrulli showed high genetic diversity and significant genetic structure, along with widespread and prevalent clones. Genetically similar isolates were aggregated within fields at a fine scale, indicating local splash dispersal of spores. Of the genotyped isolates, 132 were selected and tested for sensitivity to four fungicides commonly used in GSB management: azoxystrobin, tebuconazole, boscalid, and fluopyram. Diverse responses were discovered; however, the fungicide resistance profiles of isolates were not associated with the genetic similarity of S. citrulli isolates. All S. caricae isolates were resistant to tebuconazole, while all the S. citrulli isolates were sensitive to this fungicide. Sequencing of the target gene Cyp51 and quantification of the expression of Cyp51 and ScAtrG (an ABC transporter) by real-time PCR did not reveal the molecular mechanisms of tebuconazole resistance in S. caricae. In addition, the mating-type locus (MAT1) that controls sexual reproduction of filamentous Ascomycota was identified from the draft genomes of the three species of GSB fungi. Both mating-type idiomorphs were found within each species supporting homothallism, or self-compatibility, of all three Stagonosporopsis species causing GSB. Positive selection was detected for the genes within the MAT1 locus, which may have contributed to divergence among the GSB species. This project provides much needed information on the biology of GSB fungi, particularly on the population biology, mating system, and variation in fungicide resistance profiles, which helps us to better understand epidemics and provide suggestions on better disease management of GSB of cucurbits in the southeastern U.S.