Stemphylium lycopersici is an important pathogen causing leaf spot and other diseases of tomatoes worldwide. Although much information is available about the pathogen, little is known about dynamics of S. lycopersici in tomato fields. Seventy-nine symptomatic leaf samples were collected from two tomato cultivars grown in a farm. Fungal species associated with the disease were isolated on potato dextrose agar. All isolates were identified as S. lycopersici based on sequence analysis of combined dataset of the internal transcribed spacer (ITS) and glyceraldehyde-3-phosphate dehydrogenase (gpd) regions. The 79 isolates obtained from the two cultivars were subjected to amplified fragment length polymorphism (AFLP) analysis using three primer combinations. The Stemphylium population from the two cultivars was found to have a very low level of genetic diversity (H = 0.0948). Cluster analysis showed intermixing of isolates from the two cultivars. In addition, analysis of molecular variance (AMOVA) showed the presence of a very low level of genetic differentiation between populations obtained from the two cultivars (Fst = 0.0206). These findings indicate the presence of a high rate of gene flow between the two populations and may suggest that the two populations originated from the same inoculum source. The implications of these findings on the management of Stemphyllium-induced leaf spot of tomatoes are discussed.