Technical Abstract: Eighty years ago, Alexander Fleming described the antibiotic effects of a fungus that had contaminated his bacterial culture, kick starting the antimicrobial revolution. The fungus was later ascribed to a globally distributed asexual species, Penicillium chrysogenum. Recently, the species has been shown to be genetically diverse, and possess mating-type genes. Here, we use phylogenetic and population genetic tools to show that one of the most common Penicillium species found in air, Penicillium flemingii sp nov, Fleming’s fungus, is distinct from several other similar species including P. chrysogenum sensu stricto, P. chainii sp nov., and P. floreyi sp nov. We found each species in air and dust samples collected in and around St Mary’s Hospital where Fleming worked. Genotyping of 30 markers across the genome showed that preserved fungal material from Fleming’s laboratory was nearly identical to derived strains currently in culture collections and that a later wild progenitor strain of current penicillin producing industrial strains is also P. flemingii rather than P. chrysogenum. Global samples of P. flemingii and P. chrysogenum were found to possess mating-type genes, in a near 1:1 ratio, and show evidence of recombination, with no geographic population subdivision evident. However, no hybridization was detected between the species despite an estimated time of divergence of less than 1 MYA. Results highlight that fungal diversity is still under-recognized even in the most common groups, the value of phylogenetic species recognition, and that fungi show the potential for speciation despite complete overlap in their distribution across all scales.