Surveys during five consecutive years in four main seed potato-growing areas of Tunisia revealed large differences in Potato virus Y (PVY) incidence. Infection rates at harvest ranged from up to 19% in Cap Bon and in Jendouba, to 31% in Kairouan and 48% in Manouba. However, infection rates were very low across the country in 2003 (1-5%), except in Kairouan, and rather low in 2004 (4-10%). Secondary infections in plants growing from imported seed potatoes were assessed to be 1-6% depending on region and year. Serological analysis of a first set of 90 samples collected in 2004-2005 revealed dominance of the PVYN group (90% of the total number of PVY positives). A second set of 44 isolates from samples collected in 2006 was further analyzed with a combination of serotyping, indexing on tobacco, and RT-PCR tests targeting four genomic regions (5'Ntr/P1, HcPro/P3, CI/NIa, CP). Thirty-five of these 44 isolates were typical PVYNTN isolates, with three recombination junctions in HcPro/P3, CI/NIa, and CP regions, respectively, whereas no recombination junction was identified in the genome of five isolates belonging to the PVYN group. One additional recombinant PVYNTN isolate was recovered from a mixed infection with a PVYO isolate. Only three PVYO isolates were recovered from the samples analyzed.