Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi Syd & P. Syd, is the main disease that affects soybean crop in South America. Disease control is based on the use of site-specific fungicides (QoIs, DMIs and SDHIs), multisite fungicides and host-free period. Continuous and intensive use of fungicides has selected isolates of P. pachyrhizi, with reduced sensitivity to DMIs, QoIs and SDHIs. This work includes sensitivity studies for prothioconazole and SDHIs and genetic analysis of target site mutations in P. pachyrhizi related to sensitivity reduction to DMIs, QoIs and SDHIs collected from different regions of soybean production areas. Different CYP51 genotypes with distinct point mutations known to influence DMI sensitivity are present in Brazilian isolates. Combined mutation F120L + Y131H on CYP51 gene was the most frequent among Brazilian isolates. Mutant isolates showed higher EC50 values to the DMI prothioconazole compared to wild type isolates. Regarding QoI, almost all Brazilian isolates carried the mutation F129L in the CYTB gene. Wild type of the SdhC gene was the most frequent genotype to SDHI fungicides, but the mutation leading to the I86F amino acid exchange has also been detected. Such isolates showed higher EC50 values to the SDHIs bixafen, benzovindiflupyr and fluxapyroxad compared to wild type isolates. The most frequent genotype in our collection presented target site mutations in the CYP51 and CYTB genes. A monouredinial isolate with mutations in all three target genes was detected and is here described for the first time. Its current and further spread, and the impact on field performance of fungicides with these modes of action needs further evaluation. Display omitted •Multiple resistance Phakopsora pachyrhizi monouredinial isolate is first described.•Mutation F120L + Y131H on the CYP51 gene was the most frequent.•90% of the samples carried CYTB F129L alleles.•Wild type of the SdhC gene were the most frequent genotype to SDHI fungicides.