In Tunisia, the local germplasm of wild Gladiolus have been currently damaged by severe genetic erosion, pollution, urbanization and bad farming practices. The aim of the present study was to prospect, collect and assess morphologically 15 accessions of this species from several areas in Tunisia. The identification and characterization of 300 individuals (20 samples per each accession), was performed utilizing 20 morphological traits. In this study, morphological traits showed a large variability for wild Gladiolus species. Cluster analysis revealed two major clusters: the first includes the accessions with populations of Gladiolus italicus, the second consists of all the rest, the populations with seeds wing. The Principal Component Analysis of the measured morphological characters revealed that the 3 primary components included 70.5% of total variability. The diversity is currently threatened by genetic erosion. The sustainability of Gladiolus in Tunisia is necessary using a rapid program of conservation in situ and ex situ.