As the largest group of plant resistance genes, the nucleotide-binding site-leucine-rich repeat (NBS-LRR)genes play a critical role in plant disease defense system. The bottle gourd is a distinctive horticultural crop of the Cucurbitaceae family and powdery mildew is one of the major diseases jeopardizing crop production and quality. In this study, we have identified 84 NBS-LRR genes and divided into seven subfamilies. Phylogenetic tree and motif analysis grouped these genes into two major clusters with toll/interleukin-1 like receptor (TIR)domain or a coiled-coil domain. Subsequently, duplication events, motif sequence, orthologous gene cluster, protein-protein interaction, cis-elements were analyzed. 12 pairs of tandem duplicated genes and two pairs of segmental duplicated genes were detected in NBS-LRR family. 14 singleton genes and 62 functional cis-elements were found. The structure of 27 proteins were constructed by homology modeling and divided into five different types. 34 differentially expressed genes of NBS-LRR genes were found in PM-resistant and PM-susceptible line. Expression of NBS-LRR genes were validated by qRT-PCR under PM stress and gene Lsi04g015960 with powdery mildew8 (RPW8)domain was significantly up-regulated in PM-resistant variety. Our study provides fundamental insights of NBS-LRR family genes in bottle gourd and find a potential candidate gene Lsi04g015960 for PM tolerance breeding.