Isolates of Pyrenochaeta lycopersici, the causal agent of corky root rot on tomato plants, were assessed for physiological and genetic characteristics using conventional and molecular techniques. All isolates were able to produce necrosis on tomato roots and classified into temperature group according to the optimal growth temperatures. Specific-PCR assays and DNA sequence analysis of the ribosomal DNA (rDNA) internal transcribed spacer region confirmed the existence of both types (Type 1 and Type 2) of the pathogen among the isolates tested. All isolates were identified as Type 2 except for isolate Pl-4, which was classified as Type 1. Restriction fragment length polymorphism (RFLP) analysis with six enzymes resulted in three distinct banding patterns among the isolates depending on the length and restriction profiles of the rDNA intergenic spacer region. Inter-simple sequence-repeat analysis revealed a high level of genetic diversity among the isolates in agreement with the data of RFLP analysis. These results indicated that there were three different intraspecific groups among Turkish isolates of P. lycopersici. The presented study is the first attempting to characterize Turkish isolates of P. lycopersici. The results obtained will be useful in screening of tomato seedlings for resistance to P. lycopersici.