Tomato yellow leaf curl disease is a devastating disease of tomato (Solanum lycopersicum), which is caused by begomoviruses generally referred to as tomato yellow leaf curl virus (TYLCV). The breeding for TYLCV resistance has been based on the introgression of the Ty-3 resistance locus. Knowledge about the exact location of the Ty-3 on tomato chromosome 6 is needed to understand the genomic organization of the Ty-3 locus. In this study, we conducted a genetic analysis using a segregating population derived from a cross between resistant accession S. lycopersicum “A45” and susceptible accession S. lycopersicum “A39”. The F1 plants showed resistance to TYLCV and F1 was self-pollinated to produce F2 progeny. To screen the TYLCV resistance in 145 F2 plants, a leaf agroinfiltration method was used. F2 plants showed a classical Mendelian seregation (106 resistance : 39 susceptibility) for resistance to TYLCV respectively. SCAR and CAPS markers linked to the Ty-3 were tested for genotyping F2 plants and .genotyping and agroinfiltration results were cosegregated in the newly developed F2 population.