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Background: There are eighteen species within the <i>Penicillium</i> genus section <i>chrysogena</i>, including the original penicillin producers <i>Penicillium notatum</i> (Fleming strain) and <i>Penicillium chrysogenum</i> NRRL 1951. Other wild type isolates of the <i>Penicillium</i> genus are relevant for the production of useful proteins and primary or secondary metabolites. The aim of this article is to characterize strain specific genes and those genes which are involved in secondary metabolite biosynthesis, particularly the mutations that have been introduced during the β-lactams strain improvement programs. Results: The available genomes of several classical and novel <i>P. chrysogenum</i> strains have been compared. The first genome sequenced was that of the reference strain <i>P. chrysogenum</i> Wis54-1255, which derives from the wild type <i>P. chrysogenum</i> NRRL 1951; its genome size is 32.19 Mb and it encodes 12,943 proteins. Four chromosomes were resolved in <i>P. chrysogenum</i> and <i>P. notatum</i> by pulse field gel electrophoresis. The genomes of three industrial strains have a similar size but contain gene duplications and truncations; the penicillin gene cluster copy number ranges from one in the wild type to twelve in the <i>P. chrysogenum</i> ASP-E1 industrial strain and is organized in head to tail tandem repeats. The genomes of two new strains, <i>P. chrysogenum</i> KF-25, a producer of antifungal proteins isolated from a soil sample, and <i>P. chrysogenum</i> HKF2, a strain with carbohydrate-converting activities isolated from a sludge treatment plant, showed strain specific genes. Conclusions: The overall comparison of all available <i>P. chrysogenum</i> genomes indicates that there are a significant number of strain-specific genes, mutations of structural and regulatory genes, gene cluster duplications and DNA fragment translocations. This information provides important leads to improve the biosynthesis of enzymes, antifungal agents, prebiotics or different types of secondary metabolites.