Quinone-outside inhibitor (QoI) fungicides are effective tools for preharvest control of brown rot of stone fruit. These fungicides have a very specific site of action so the risk of resistance selection is high. The sensitivity of Monilinia fructicola (G. Winter) Honey isolates to azoxystrobin (QoI) was investigated in 143 isolates collected between 2002 and 2011 from four Brazilian states in orchards with different frequencies of fungicide use (0 to 6 fungicides sprays/season). Sensitivity of the isolates to azoxystrobin was determined in vitro, by inhibition of mycelial growth and spore germination on fungicide-amended media or ex vivo by pathogen inoculation in untreated or treated fruit with azoxystrobin. Potential mutations in codons 143, 137, and 129 of the cytochrome b (Cyt b) gene and the occurrence of an intron immediately after codon 143 were analyzed in a subpopulation of the isolates. The M. fructicola population of São Paulo State was less sensitive to the fungicide than the population from the states of Paraná, Santa Catarina, and Rio Grande do Sul. The low sensitivity of the isolates was confirmed also by comparing to the sensitivity of the baseline isolates. Mutations in G143A, F129L, and G137R in Cyt b gene were not found. In addition, 58 isolates tested showed an intron after codon 143 in Cyt b gene. Our results indicate that other mechanisms of selection for low sensitivity to QoI fungicides should be investigated.