The bottle gourd or calabash [Lagenaria siceraria (Mol.) Standl.] (2n =2x=22), also known as opo squash or long melon, is an important cucurbit crop worldwide. Commonly used as a vegetable, bottle gourd fruits have a peculiar umami taste that is important for their market value. To date, the umami chemical ingredients of bottle gourd fruits and their genetic determinants remain unidentified. In the current study, the relationship between umami flavor and free amino acid (FAA) contents was analyzed using a representative panel of twenty-eight accessions. Among the FAAs identified, free glutamate (Glu) was found to have the most significant association with umami taste, suggesting that free Glu is the main umami-conferring ingredient of bottle gourd. To further identify the genetic regions associated with umami taste, a genome-wide association study (GWAS) for free Glu content was conducted in a diversity panel consisting of 139 inbred lines, each sequenced with a restriction siteassociated DNA sequencing (RAD-Seq) technology. Seventeen SNPs were identified as associated with free Glu content, each accounting for 7.5% to 15.1% of the phenotypic variation. The number of the favored alleles for each line varied from 0 to 13. Free Glu content exhibited a significant positive correlation with the number of the favored alleles. These results indicated that there is considerable genetic potential to develop new varieties with stronger umami taste through targeting more favored alleles of free Glu content. To our knowledge, this is the first GWAS study in bottle gourd. Our work reveals the genetic architecture of umami taste in the bottle gourd and will help facilitate molecular breeding of cultivars with improved flavor.