Botrytis cinerea, considered for a long time as a generalist fungal pathogen of a multitude of plants, was recently shown to exhibit significant population structure in France according to the host, suggesting sympatric specialization. Recent models also showed that adaptation to new hosts may facilitate the process of sympatric speciation in fungal plant pathogens. The present work aimed at investigating if host plants, combined with geographic origin and growing systems, shape the diversity and structure of Tunisian populations of B. cinerea. We genotyped 153 isolates with 9 microsatellites. In all the investigated populations, the fungus reproduced mainly sexually. Gene flow was significantly reduced between greenhouses and open fields from strawberry but not from grapevine. Populations from tomatoes, sampled under greenhouses only, exhibited a low genotypic diversity. The effects of plant and geography from open fields were investigated on a sample of 74 isolates. Six populations were inferred, mainly structured according to a geographic barrier corresponding to the Grande Dorsale Mountain. However, this effect could not be separated from the host plant origin of isolates. The analysis of 63 isolates recovered from strawberries and faba beans in the Cap Bon and Centre regions did not reveal any significant effect of plant on pathogen population differentiation.