Body color is an interesting economic trait in fish. Red tilapia with red blotches may decrease its commercial values. Conventional selection of pure red color lines is a time-consuming and labor-intensive process. To accelerate selection of pure lines through marker-assisted selection, in this study, double-digest restriction site-associated DNA sequencing (ddRAD-seq) technology was applied to genotype a full-sib mapping family of Malaysia red tilapia (Oreochromis spp.) (N = 192). Genome-wide significant quantitative trait locus (QTL)-controlling red blotches were mapped onto two chromosomes (chrLG5 and chrLG15) explaining 9.7% and 8.2% of phenotypic variances by a genome-wide association study (GWAS) and linkage-based QTL mapping. Six SNPs from the chromosome chrLG5 (four), chrLG15 (one), and unplaced supercontig GL831288-1 (one) were significantly associated to the red blotch trait in GWAS analysis. We developed nine microsatellite markers and validated significant correlations between genotypes and blotch data (p
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