In this study, we investigated the pathogenicity and patulin production by ten strains of Penicillium expansum on various fruits (apples, apricots, kiwis, plums and peaches) at two (4°C and 25°C) different temperature regimes. All strains caused the infectious rots on all fruits at 4 and 25°C except one strain (PEX 09) at 4°C. Two strains (PEX 20 and PEX 12) out of ten produced the highest amounts of patulin on all fruits tested. The patulin production by P. expansum is high at 25°C compared to 4°C. All strains of P. expansum accumulated patulin ranging from 100-13,200 μg/kg and nine strains ranging from 100-12,100 μg/kg in all fruits at 25°C and 4°C, respectively. Among ten strains of P. expansum, strain PEX 20 produced the greatest amount of patulin on apricots (13,200 μg/kg of rotten fruit) and on apples (12,500 μg/kg) at 25°C after 9 days of incubation. At 4°C, this strain produced 12,100, 12,000, 2,100 and 1,200 μg/kg of patulin on apricots, apples, plums and peaches, respectively, after 45 days of incubation. Strain PEX 12 produced the highest amount of patulin on kiwis (10,700 μg/kg) at 25°C and 10,300 μg/kg at 4°C. Patulin production by P. expansum on peaches and plums at both temperatures were lower than other fruits. The results of this study showed that careful removal of rotten fruits is essential to produce patulin-free fruit juice, since high patulin levels in apricots, apples and kiwis could result in a level greater than 50 μg/kg of this mycotoxin in finished fruit juices, when one contaminated fruit occurs in 264, 250 and 214 fruits, respectively. So, the fruit processors should take care in not using rotten fruits for juice production to avoid the patulin problem worldwide, since this study proved that most important fruits being used for juice production and direct human consumption are susceptible to P. expansum and subsequent patulin production even at low temperatures. This is the first comprehensive report regarding patulin production by different strains of P. expansum on various fruits from Italy at different temperature regimes.