Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder inherited as an autosomal recessive trait. MLD is caused by the deficiency of arylsulfatase A (ARSA), a lysosomal enzyme that catalyzes the first step in the degradation of sulfated glycolipids, which are essential components of the myelin sheet. Notably, between 7% and 15% of healthy individuals show in vitro deficiency of ARSA, a condition called ARSA pseudodeficiency (ARSA-PD). To date, 151 ARSA-MLD mutations have been reported in the gene encoding ARSA (ARSA), among which IVS2+1G>A and P426L occur at high frequencies in most of the studied populations. The aim of this work was to identify ARSA mutant alleles in a cohort of 27 unrelated Brazilian MLD patients. The most frequent ARSA-MLD mutation, IVS2+1G>A, and the ARSA-PD polymorphisms, N350S and 1524+95A>G, were detected using real-time PCR, while the remaining mutations were detected using direct sequencing of ARSA. In concordance with previous reports, IVS2+1G>A and P426L were the most common ARSA-MLD mutations in our cohort of MLD patients, found at frequencies of 0.05 and 0.08, respectively. Interestingly, two mutations previously reported as rare, 103_110del8 and 1190_1191insC, were found at higher frequencies in our cohort of MLD patients, 0.08 and 0.06, respectively. Additionally, 11 other rare ARSA-MLD mutations were found at lower frequencies in our cohort of MLD patients. To our knowledge, this is the first systematic genotypic characterization of MLD patients from Latin America. This work highlights the genetic heterogeneity of MLD, and supports genotype-phenotype associations, which become more important as specific treatments are being developed for this devastating disorder.