In the framework of a Collaboration Agreement between CREA and ARSIAL, a morpho-phenological, chemical, and genetic characterization of maize populations native to the Lazio region was carried out. During 2022 and 2023, a set of 50 accessions, belonging both to ARSIAL and CREA maize collections, were multiplied in Bergamo. Morpho-phenological descriptors were recorded in the field: plant height, ear height, and male and female flowering time. The grain chemical composition in terms of protein, lipid, starch, ash and fiber was evaluated by near-infrared spectroscopy (NIRS). A double-digest restriction-site-associated DNA sequencing (ddRADseq) strategy was used to genotype the landraces. The two collections were not significantly different in terms of grain chemical composition. On the other hand, the ARSIAL and CREA germplasm showed a different distribution in the three cluster-based population structure obtained by ddRADseq, which largely corresponded to the distribution map of their collection sites. The materials from the Lazio region maintained by ARSIAL and CREA were revealed to be different. The comparison between the two groups of landraces showed the importance of characterizing germplasm collections to promote the recovery and valorization of local biodiversity.