Using molecular analysis has helped to accurate identify plant pathogens and determining variation among their isolates. We have used molecular methods to identify species and variations among their isolates of pathogens causing cucurbit powdery mildew, Phytophthora blight of peppers and cucurbits, and internal discoloration of horseradish roots. Several other studies on determining species of fungi (e.g., Fusarium and Phytophthora) have also been published. After sequencing the internal transcribed spacer of nuclear ribosomal DNA (nrDNA) region of 119 isolates of powdery mildew fungi collected from six species of cucurbits in the United States, Italy, and Chile, Podosphaera xanthii was found the only species causing powdery mildew of cucurbits. Genetic variation among the isolates showed significant clustering trend of isolates. Random amplified polymorphic DNA markers were employed to assess genetic variation among 24 isolates of Phytophthora capsici in Illinois. Mean pair group analysis clustered isolates into six distinct groups. Original study showed that Verticillium dahlia caused internal discoloration of horseradish roots. Using molecular methods, it was revealed that the internal discoloration of horseradish roots is a disease complex and can be caused by one or any combination of five fungal species, including V. dahlia, V. longisporum, Fusarium solani, F. oxysporum, and F. commune. “Die Fusarien,” published in 1935, described 65 species, 55 varieties, and 22 forms of Fusarium. Using morphological characteristics, number of species of Fusarium was reduced to nine in 1945. The application of phylogenic species concept based on the DNA sequencing resulted in describing 70 species of Fusarium in 2006.