Bitterness in melon (Cucumis melo L.) seriously affects fruit quality and marketability. The present study aimed to search for the molecular basis of bitterness in melon based on positive genetic strategy. An F2 segregating population was constructed using 100 individuals derived from a cross between the non-bitter female parent C69 and the bitter male parent C14. A genetic linkage map was constructed based on 2b-RAD sequencing, which was then combined with the phenotypic trait of fruit bitterness for QTL mapping and genome-wide association study, and a PAV was detected by the in-depth information of 2b-RAD sequencing. Key genes controlling fruit bitterness were determined by re-sequencing of the two parents. The F1 population showed strong bitterness in fruit, and the F2 population had 81 and 19 individuals with bitter and non-bitter fruit respectively, which approximated the 3:1 segregation ratio (?2=1.92，P=0.1659). A linkage map was constructed based on 477 SNPs distributed in 10 linkage groups. The total length of the linkage map was 337.79 cM, with an average distance of 0.71 cM between adjacent markers. Whole genome QTL mapping identified one QTL for bitterness, which explained 20% of the phenotypic variation on LG08 (corresponding to chromosome 9). Genome-wide association study detected seven SNPs significantly associated with bitterness in melon, which were located on the target QTL region for bitterness on chromosome 9. PAV analysis detected the presence or absence of a long fragment (from 21,707,702 bp to 21,743,072 bp on chromosome 9) in the QTL region, which explained the fruit bitterness or the lack of it in F2 population. The in-depth re-sequencing of the two parents were checked and it was found that the PAV region was larger, of about 62 kb, and was involved in nine consecutive genes (MELO3C005601, MELO3C005602, MELO3C005603, MELO3C005604, MELO3C005605, MELO3C005606, MELO3C005607, MELO3C005608 and MELO3C005609), of which five were cytochrome CYP450 genes. The constructed phylogenetic tree revealed that these five cytochrome P450 genes were in the same clade with the cytochrome P450 gene clusters CYP81Q58, CYP81Q59 and CYP712D8 involved in cucurbitacin C/B/E synthesis, and may function similarly. The phenotypes of F1 and F2 populations indicated that the fruit bitterness was controlled by one dominant gene in melon line C14. The PAV located in the QTL region co-segregated with the bitterness phenotype and explained 100% phenotypic variation in the F2 population, which suggested that the absence of the nine genes was the main cause for the non-bitterness of C69. The five cytochrome CYP450 genes are potentially part of the gene cluster of CuB biosynthesis pathway. Comparative analysis identified two bHLH transcriptional factors tightly linked with PAV in present study, which controlled the formation of CuB, CmBr (MELO3C005610) and CmBt (MELO3C005611) located in the consecutively linked regions in chromosome 9. The present results provide new theoretical support and target of molecular marker assisted breeding of melon.