Monilinia fructicola causes considerable damage to cultivated stone fruits in the temperate regions with an important economic impact. Monitoring the strains resistant to fungicides is essential to reduce economic losses associated with the peach and nectarine market. Although several works have focused on benzimidazole fungicide resistance worldwide in Monilinia spp., limited data report the benzimidazole resistance in European M. fructicola isolates. In order to assay the development of resistance to thiophanate methyl, the Alamar Blue test, a quick and reliable assay, was used and the results compared with those obtained with conventional amended medium. Our results show for the first time the presence in Italian M. fructicola isolates of a phenotype resistant to thiophanate methyl. In particular, 46 out of 63 isolates were found resistant, with EC50 values ranging from 0.99 μg ml-1 to 57.59 μg ml-1, values equal or higher than the inhibitory dose (1 μg ml-1). Point mutations in the β-tubulin gene were analyzed in 18 representative M. fructicola isolates, 15 with different levels of resistance (low and high resistance) and three sensitive. All resistant isolates tested showed a point mutation at codon 198 with respect to sensitive isolates isolates, i.e. GCA instead of GAA. In addition, all Italian isolates revealed a point mutation at codon 83 in the β-tubulin gene where the arginine was converted to glutamine with a punctual allelic change CAA instead of CGA.