Symptoms of clover rot caused by Sclerotinia trifoliorum or S. sclerotiorum are identical, making differentiation and identification of the causal species difficult and time consuming. Polymerase chain reaction (PCR) amplification and nucleotide sequencing were used to examine 40 isolates of S. trifoliorum (29 from Poland, 11 from the United States) and 55 isolates of S. sclerotiorum (26 from Poland, 29 from the United States). We determined that amplification of the β-tubulin and calmodulin genes with TU1/TU2/TU3 and SscadF1/SscadR1 PCR primers and the presence of introns and single-nucleotide polymorphisms (SNP) within the ribosomal DNA (rDNA) as detected with NS1/NS8 and internal transcribed spacer (ITS)1/ITS4 PCR primers are effective for rapidly and accurately differentiating between the two species of Sclerotinia. In addition, our research revealed a lack of intraspecies variation within S. sclerotiorum isolates from the United States and Poland using these same molecular markers. We detected a relatively high degree of intraspecies variability among isolates of S. trifoliorum from the United States and Poland using the presence of introns and SNP within the rDNA. SNP and nuclear small-subunit rDNA analyses revealed distinct groups of S. trifoliorum among the isolates used in this study. The results of this study provide useful information for clover breeders and pathologists looking to develop clover varieties with durable resistance.