AbstractA combination of two approaches, namely QTL analysis and gene enrichment analysis were used to identify candidate genes in the “QTL-hotspot” region for drought tolerance present on the Ca4 pseudomolecule in chickpea. In the first approach, a high-density bin map was developed using 53,223 single nucleotide polymorphisms (SNPs) identified in the recombinant inbred line (RIL) population of ICC 4958 (drought tolerant) and ICC 1882 (drought sensitive) cross. QTL analysis using recombination bins as markers along with the phenotyping data for 17 drought tolerance related traits obtained over 1–5 seasons and 1–5 locations split the “QTL-hotspot” region into two subregions namely “QTL-hotspot_a” (15 genes) and “QTL-hotspot_b” (11 genes). In the second approach, gene enrichment analysis using significant marker trait associations based on SNPs from the Ca4 pseudomolecule with the above mentioned phenotyping data and the candidate genes from the refined “QTL-hotspot” region showed enrichment for 23 genes. Twelve genes were found common in both approaches. Functional validation using quantitative real-time PCR (qRT-PCR) indicated four promising candidate genes having functional implications on the effect of “QTL-hotspot” for drought tolerance in chickpea.