Bitter gourd, Momordica charantia L., is a vegetable with one of the highest nutrition values in the world. Despite this, limited research has been conducted on bitter gourd cultivars; thus, the efficient use of genetic resources for bitter gourd improvement remains a challenge. Here, we performed restriction-site associated DNA (RAD) sequencing for 133 bitter gourd cultivars, including three types of bitter gourd (Smooth, Pearl, and Dading bitter gourd). We found that the genetic diversity of bitter gourd cultivars was very narrow with nucleotide diversity (π) values of 6.79 × 10^− 4 (Pearl), 6.06 × 10^− 4 (Smooth), and 3.99 × 10^− 4 (Dading). It is possible that the commercial cultivars have almost exhausted the genetic diversity of bitter gourd resources in China. We found that Smooth and Pearl cultivars had more mixed genetic backgrounds, whereas Dading cultivars were more distinct. In addition, we had a try to use this cultivar population to conduct GWAS on seed coat color and identified one significant determined region that spanned ~ 300 kb on chromosome 3. The results of this study will help to broaden our capacity to monitor the genetic diversity of bitter gourd cultivars.