Bitter gourd is a traditional melon vegetable in China, characterized by a very narrow genetic background of variety resources. Consequently, there is an urgent need to broaden the utilization of germplasm resources in breeding applications. In this study, we used the small-fruited wild subspecies Momordica charantia ssp. macroloba ‘NJ’ as the maternal parent and the common subspecies M. charantia ssp. charantia ‘Tan’ as the paternal parent. Based on resequencing and bin marker genotyping, we constructed a genetic map of inter-subspecies in bitter gourd, and employed the multiple-QTL model (MQM) method to map quantitative trait loci (QTL) associated with fruit-related traits. The results showed that the constructed genetic map of inter-subspecies in bitter gourd comprised 1,563 bin markers, spanning 11 chromosomes with a total genetic distance of 1,556.91 cM and an average marker interval of 1.00 cM. A comparison of the genetic and physical maps revealed an inversion region of approximately 11 Mb on chromosome MC08 between the two subspecies of bitter gourd. The QTL mapping results indicated that a total of seven QTL reached the LOD detection threshold, including two for fruit length (qFL5.1 and qFL7.1), two for fruit shape index (qFSI1.1 and qFSI4.1), two for fruit weight (qFW5.1 and qFW6.1), and one for fruit shape (qFS5.1). The fruit length QTL qFL5.1, fruit shape QTL qFS5.1, and fruit weight QTL qFW5.1 colocalized to form a major-effect QTL interval, with contribution rates of 23.00%, 30.20%, and 24.80%, respectively. The two QTLs for fruit shape index (qFSI1.1 and qFSI4.1) were different from the fruit shape QTL qFS5.1, with contribution rates of 22.80% and 14.60%, respectively. The findings of this study contribute to our understanding of the genomic variation between the two subspecies of bitter gourd and provide the foundation for inter-subspecific germplasm innovation and gene mining of fruit-related traits.