Isolates of Verticillium fungicola, collected over a period of 45 yr from various geographical sites, were analysed for their responses to four fungicides (benomyl, thiabendazole, chlorothalonil and diethofencarb), and for colony morphology, virulence and DNA polymorphisms. Three different colony morphologies were observed for this population of isolates including fluffy, dense and appressed. The majority of isolates had the appressed colony morphology. Most isolates exhibited high levels of virulence. The appearance in this population of both benomyl and thiabendazole resistance was correlated with the introduction of benomyl for use on mushrooms. Eighty-eight percent of the isolates were cross-resistant between benomyl and thiabendazole and 80% exhibited negatively correlated cross-resistance between benomyl and diethofencarb. Seventy percent of the isolates exhibited negatively correlated cross-resistance between thiabendazole and diethofencarb. The level of resistance to chlorothalonil was relatively high even prior to its introduction and overall the response to this fungicide appears to have varied little over the last 45 yr. DNA polymorphisms, as detected by randomly amplified polymorphic DNA (RAPD) analysis, divided the isolates into four groupings. Limited correlations could be drawn between RAPD groupings, fungicide response and geographical origin. All recently collected isolates (1993–5) were extremely similar in their response to the four fungicides and had appressed colony morphology, high virulence and were members of RAPD group four. The lack of variation in the recent isolates as compared to the older isolates indicates that the V. fungicola population may be becoming more homogeneous.