There are many species of Narcissus in diverse areas of the world in natural or cultured form and there is no complete information about their genetic status, especially the relatedness within a species. Thus, the current study applied intersimple sequence repeat (ISSR) markers to estimate the genetic diversity of 31 accessions, including 30 accessions of Narcissus tazetta, collected from 16 regions of Iran and one known exotic narcissus species that is being cultivated in Iran, and identification of tolerant genotypes for deficit irrigation by evaluation of their morpho-physiological characteristics. Seventeen anchored ISSR primers from a total of 19 tested ISSR primer pairs were used and produced 206 bands of different sizes. The average percentage of polymorphic bands was 96.02%. The maximum resolving power (8.32), polymorphic information content average (0.44), and marker index values (5.61) were observed for the primers of 811, 828, and 811, respectively. The unweighted pair group method with arithmetic mean based on Jaccard’s coefficients was used to assign the genotypes to one of two major clusters. Both clusters were divided into two subclusters, with single and double flowers separating into subgroups. The results showed that ISSR markers can be used as a diagnostic tool to evaluate genetic variation in Narcissus genotypes and reveal their relationships. The results of screening study identified drought-tolerant accessions. They were clustered into two major groups: drought-tolerant accessions with single flowers and drought-sensitive accessions having double and semidouble flowers. The findings presented can be used in breeding programs for different Narcissus genotypes.