Penicillium chrysogenum is a ubiquitous airborne fungus detected in every sampled region of the Earth. Owing to its role in Alexander Fleming's serendipitous discovery of Penicillin in 1928, the fungus has generated widespread scientific interest; however its natural history is not well understood. Research has demonstrated speciation within P. chrysogenum, describing the existence of four cryptic species. To discriminate the four species, we developed protocols for species-specific diagnostic PCR directly from fungal conidia. 430 Penicillium isolates were collected to apply our rapid diagnostic tool and explore the distribution of these fungi across the London Underground rail transport system revealing significant differences between Underground lines. Phylogenetic analysis of multiple type isolates confirms that the ‘Fleming species’ should be named Penicillium rubens and that divergence of the four ‘Chrysogenum complex’ fungi occurred about 0.75 million yr ago. Finally, the formal naming of two new species, Penicillium floreyi and Penicillium chainii, is performed.