A mycological survey of fungi, present in several stages of the manufacturing of cork discs for champagne stoppers in Portugal, was made. Sixty-nine strains belonging to the Glabra series of the genus Penicillium were isolated and subsequently grouped according to their partial β-tubulin gene sequences. Six groups with different partial β-tubulin gene sequences were observed, and a selection of isolates of each group was made. These selected isolates and various related ex-type strains were subjected to a taxonomical study using a polyphasic approach. This approach included analysis of macro- and microscopic features, the comparison of extrolite profiles and sequencing a part of the β-tubulin and calmodulin gene. The six β-tubulin types were reduced to three different species. One group of isolates was centred on the ex-type strain of P. glabrum, a second group accommodated the type strain of P. spinulosum and a third group contained isolates which were unique in their β-tubulin and calmodulin sequences, extrolite profiles and growth characteristics. This group of isolates is described as the new species Penicillium subericola. The type strain of P. subericola CBS 125096T was isolated from Portuguese raw cork, but additional isolates were found from soil, air and lumen.