Soybean target spot, caused by the fungus Corynespora cassiicola, is an important disease in northwestern Argentina (NWA). A cultural, morphological, and virulence characterization of 24 C. cassiicola isolates from different geographic localities in NWA was performed. The isolates were classified into five different cultural phenotypes, one of which has not been previously described. The ribosomal DNA internal transcribed spacer region sequence from five representative isolates (587 bp) exhibited 100% identity with C. cassiicola. A high variability of conidial morphology was observed among isolates, but no correlation with cultural phenotype was observed. When the soybean cultivar A 8000 RG was challenged with the 24 isolates, different degrees of virulence were observed, ranging from highly virulent to nonvirulent. No association of virulence with cultural or morphological characters was observed, but a relationship with geographical origin was demonstrated. Histopathological studies were performed on a nonvirulent and a highly virulent isolate. Microscopic observations of infected tissues of the former showed low mycelium development restricted to the upper epidermis, a thickening of the cuticle and primary wall of subepidermal cells, and accumulation of callose in phloematic vessels. In tissue infected with the latter, there was abundant mycelium development accompanied by cellular disorganization in mesophyll cells. Pathological challenges of isolates on nine different cultivars indicated that the degree of virulence of isolates depends on the plant genotype, demonstrating that the C. cassiicola–soybean interaction is highly specific. Understanding the genetic basis of this interaction will provide new information for better disease management and breeding strategies.