Gladiolus is considered one of the most significant globally ornamental crops, extensively utilized as a cut flower and for outdoor landscaping. However, the local destruction of Iranian Gladiolus as a weed threatens the biodiversity of this species. This research aimed to compare Iranian Gladiolus populations. A total of 76 Iranian Gladiolus accessions were collected. These accessions exhibited significant phenotypic variability across all the studied traits. Geographical factors were found to significantly contribute to this diversity. Additionally, to explore the association between genetic markers and phenotypic traits, both ISSR and IRAP markers were employed. Significant correlations were identified between stem length and latitude (r = 0.746, p < 0.01). Moreover, floret number showed a strong correlation with spike length and longitude (r = 0.777, p < 0.01 and r = 0.658, p < 0.05, respectively). The Hmdn8 population from Hamedan province exhibited superior values across all phenotypic traits. Furthermore, the Krdstn4 population from Kurdistan province, known for its superior inflorescence traits, was identified as suitable for specific breeding purposes. The climatic conditions of Kurdistan, characterized by a cold semi-humid climate and high annual rainfall, indicate that higher latitudes and colder periods are favorable for Gladiolus growth and flowering. A total of 146 polymorphic bands were produced from two types of markers, ISSR and IRAP. The association analysis revealed that the ISSR4-3, ISSR2-20 and ISSR5-24 markers showed significant correlations with stem length and floret number. Additionally, informative markers were identified for other traits, demonstrating significant associations with multiple traits in Gladiolus. These findings are crucial for identifying crucial genomic regions for Gladiolus breeding programs.