Luffa cylindrica (L.) M. Roem. is an underutilized vegetable and a potential biodiesel crop for future. Its fruits are edible and used as vegetable. To aid in the selection of lines for breeding, genetic variation and structure of ten populations of L. cylindrica were determined with three morphological characters and 23 sequence-related amplified polymorphism (SRAP) markers. All the three morphological characters were found diverse among 45 accessions. The SRAP primers produced a total of 177 bands, out of which 129 were polymorphic. Informative markers assessed by different measures (polymorphism information content = 0.18, marker index = 1.26, resolving power = 2.87) will direct the selection of primers for cultivar identification in L. cylindrica. Low level of genetic differentiation among populations and higher level within populations (G
st= 0.4073, N
m= 0.7277, H
e= 0.124, I= 0.20) was detected that might be due to cross-pollinated nature of L. cylindrica. The clustering pattern obtained through dendrogram and principal coordinate analysis was loosely concordant with the geographical distribution. The Bayesian structure analysis indicated an admixture type of population distribution. The results designate that SRAP and morphological markers are informative for characterization of L. cylindrica and identification of distinctive cultivars.