The water buffalo is among the most important livestock species of southern Asia, contributing greatly to the ecosystem and rural livelihood of the region. The identification of large-scale single nucleotide polymorphisms in this species would greatly facilitate our understanding of the genetic basis of economically important traits such as milk production, fertility traits and general health traits. The present study investigated the cost-effective method of exome capture and single nucleotide variant (SNV) identification from genomic DNA of Jaffrabadi buffalo using biotin-labelled cDNA as probes. Sequencing of enriched fragments generated 608 Mb of data, which was mapped to a Bos taurus genome assembly followed by variant calling and annotation. Furthermore, 393 coding SNVs were identified, leading to 143 non-synonymous substitutions (nsSNVs) in 75 genes. Of the 75 nsSNV-containing genes, four matched the genes that have previously been reported to be potentially associated with economically important traits such as milk production and meat production. Furthermore, functional annotation using gene ontology (GO) enrichment identified categories such as glutamate receptor activity (GO: 0008066) enriched in the fertility trait samples. These results provide a framework for the application of cost-effective methods of target capture in SNV detection from non-model organisms such as the water buffalo.