Skin biopsy samples from 145 relapse leprosy cases and from five different regions in Brazil were submitted for sequence analysis of part of the genes associated with Mycobacterium leprae drug resistance. Single nucleotide polymorphisms (SNPs) in these genes were observed in M. leprae from 4 out of 92 cases with positive amplification (4.3%) and included a case with a mutation in rpoB only, another sample with SNPs in both folP1 and rpoB, and two cases showing mutations in folP1, rpoB, and gyrA, suggesting the existence of multidrug resistance (MDR). The nature of the mutations was as reported in earlier studies, being CCC to CGC in codon 55 in folP (Pro to Arg), while in the case of rpoB, all mutations occurred at codon 531, with two being a transition of TCG to ATG (Ser to Met), one TCG to TTC (Ser to Phe), and one TCG to TTG (Ser to Leu). The two cases with mutations in gyrA changed from GCA to GTA (Ala to Val) in codon 91. The median time from cure to relapse diagnosis was 9.45 years but was significantly shorter in patients with mutations (3.26 years; P = 0.0038). More than 70% of the relapses were multibacillary, including three of the mutation-carrying cases; one MDR relapse patient was paucibacillary.