A high frequency transformation system has been developed for Penicillium paxilli using pAN7-1. Up to 44% of the primary transformants were heterokaryons. Loss of hygromycin resistance was observed in primary transformants that were sub-cultured on non-selective media, but single spores of these primary transformants were mitotically stable on both selective and non-selective media. A molecular analysis of the transformants generated showed that 78% had single-site integrations, with half of these containing a single copy of pAN7-1. CHEF-gel electrophoresis showed that P. paxilli has at least six chromosomes with a total genome size of about 23.4 Mb.