Two published genomes of Luffa cylindrica, ‘SG2019’ and ‘SO3’, were used to develop polymorphic SSR markers for L. cylindrica by searching and comparing the SSR motifs at the same site in the two genomes. Based on the SSR search conditions and primer design criteria, 2130 polymorphic SSR markers for L. cylindrica were developed. The main motif type was dinucleotide, accounting for 80.28% of the total; the main motif units were AT/AT and AAT/ATT, accounting for 87.80% of the total. Furthermore, 40 polymorphic SSR markers developed in this study were randomly selected and amplified in 24 Luffa samples. According to the results, the 40 polymorphic SSR markers showed an amplification rate of 100% and a polymorphism rate of 80%. Cluster analysis classified the 24 Luffa samples into two main groups, L. cylindrica and L. acutangula. Overall, the polymorphic SSR markers developed in this study display a high polymorphic rate and reliable utilization value.