Using controlled whitefly-mediated inoculation techniques, seven Lycopersicon chilense accessions, highly resistant to isolates of tomato yellow leaf curl virus (TYLCV) from Southern Europe, TYLCV-Sr, were selected. All exhibited similar levels of partial resistance, bring symptomless and with low levels of viral DNA accumulation. However, a differential response to infection was found in interspecific hybrids with tomato and inbred lines derived from different L. chilense accessions, allowing a precise discrimination among them. This selection procedure which considers the expression of the resistance genes in the tomato genetic background led to the selection of two highly resistant F-1 hybrids derived from L. chilense LA 1932 and LA 1938. A backcrossing programme was initiated, selecting for horticultural characteristics and TYLCV resistance, in field and controlled inoculation conditions. As a result of this programme, six advanced breeding lines (UPV Ty 1, 3, 6, 9, 17 and 53), exhibiting a high level of resistance to TYLCV-Sr, were obtained. Under high inoculum pressure conditions these lines suffered only 30-40% yield loss relative to non-infected control plants, and compared with 90-95% yield loss in susceptible controls. These lines also have horticultural characteristics appropriate for the fresh market tomato cultivation system in this area, and are a good base material for obtaining commercial hybrids highly resistant to different isolates of TYLCV.