Abstract Sugarcane brown rust, caused by Puccinia melanocephala , is a severe foliar disease that occurs in almost all countries where sugarcane is grown. The main control strategy is the use of resistant cultivars. The aim of this work was to identify molecular markers linked to genomic regions associated with a novel brown rust resistance source in sugarcane. An F 1 progeny of 300 clones was obtained from a cross between TUC 00–36 and RA 87–3, highly susceptible and highly resistant to brown rust, respectively. A total of 60 F 1 clones with extreme phenotype, either highly susceptible or highly resistant to brown rust, were selected. This “pooled tail” population was tested for reactions to brown rust under natural infection in the field during two crop seasons and under artificial infection in the greenhouse. Whole‐genome profiling was performed by DArT‐seq technology. Phenotypic data under both conditions and 23,299 single‐nucleotide polymorphisms (SNPs) obtained from genotyping were analysed to identify markers linked to the resistance trait. Single mapping analyses and subsequent multiple regression showed that 34 SNP markers were significantly linked to resistance alleles. These SNPs jointly explained 69% and 66% of the total phenotypic variation ( R 2 ) observed for field and controlled conditions, respectively. The mapping of the 34 SNP sequences revealed that 19 markers aligned to the sugarcane genome, whereas 12 markers aligned to the sorghum genome, all grouped on chromosome 5 with some functional annotations related to vegetal defence response. These marker loci could contribute to the development of molecular tools for molecular marker‐assisted breeding.