Penicillium expansum, a patulin producer fungus, is the most important fungus causing decay in cold stored both apples and pears. This can lead to patulin contaminated by-products. The aim of this assay was to evaluate the phenotypical and physiological variability in the population of P. expansum that cause fruit spoilage in post-harvest stages in Lleida (Spain). In total, 101 isolates of P. expansum from the 2004 and the 2005 seasons were obtained from decayed fruits. Significant differences were found in the observations from both seasons. Variability of the isolates in each season seemed to be partially explained by differences in growth in media, patulin accumulation and resistance to fungicides. Patulin production was detected in almost 100% of the isolates. Variability existing in P. expansum population could not be totally explained, but the above mentioned variables explained up to 74% of the diversity in some cases. The results obtained point to the existence of different populations of P. expansum in each season and may explain the differences in fungicide resistance observed between both seasons. The capacity to colonize apple flesh and some variables involved in fruit colonization were not a source of variation neither in each season nor when both seasons were compared. As storage rooms are cleaned and disinfected each season, this suggests that each season, the populations in storage rooms develop only from strains capable to colonize apple flesh. This may lead to rapid sporulation and spreading of spores.