Quinone outside inhibitor (QoI) and succinate dehydrogenase inhibitor (SdhI) fungicides are respiration inhibitors (RIs) used for preharvest control of brown rot of stone fruit. Both chemical classes are site-specific and, thus, prone to resistance development. Between 2006 and 2008, 157 isolates of Monilinia fructicola collected from multiple peach and nectarine orchards with or without RI spray history in South Carolina and Georgia were characterized based upon conidial germination and mycelial growth inhibition for their sensitivity to QoI fungicides azoxystrobin and pyraclostrobin, SdhI fungicide boscalid, and a mixture of pyraclostrobin + boscalid. There was no significant difference (P = 0.05) between EC50 values for inhibition of conidial germination versus mycelial growth. The mean EC50 values based upon mycelial growth tests for 25 isolates from an orchard without RI-spray history were 0.15, 0.06, 2.23, and 0.09 μg/ml for azoxystrobin, pyraclostrobin, boscalid, and pyraclostrobin + boscalid, respectively. The respective mean EC50 values for 76 isolates from RI-sprayed orchards in South Carolina were 0.9, 0.1, 10.7, and 0.13 μg/ml and for 56 isolates from RI-sprayed orchards in Georgia were 1.2, 0.1, 8.91, and 0.17 μg/ml. Overall, mean EC50 values of populations from RI-sprayed orchards increased three-, two-, five-, and twofold between 2006 and 2008 for azoxystrobin, pyraclostrobin, boscalid, and pyraclostrobin + boscalid, respectively. A subset of 10 M. fructicola isolates representing low and high EC50 values for azoxystrobin, boscalid, and boscalid + pyraclostrobin was selected for a detached fruit assay to determine disease incidence and severity following protective treatments of formulated RI fungicides at label rates. Brown rot incidence was greater than 50% when fruit were inoculated with isolates having EC50 values of 2, 4, and 0.6 μg/ml for azoxystrobin, boscalid, and pyraclostrobin + boscalid, respectively. Pyraclostrobin failed to control any of the isolates tested in detached fruit assays. Based on minimum inhibitory concentration and brown rot incidence data, we recommend using 3 and 0.75 μg/ml as discriminatory doses to distinguish between sensitive isolates and those with reduced sensitivity to azoxystrobin and pyraclostrobin + boscalid, respectively. Results from our in vitro and in vivo assays indicate a shift toward reduced sensitivity in M. fructicola from the southeastern United States. No cross-resistance was observed between the QoI and the SdhI fungicides, which implies that rotation or tank mixtures of these two chemical classes can be used as a resistance management strategy.