The lentil (Lens culinaris Medik.) belongs to the legume family (Fabaceae), and it possesses benefits at nutritional, sustainable, and crop rotation system levels. Its occurrence and consumption in Chile originated from imports from Canada, but molecular tools now group Canadian lentils apart from those of the Mediterranean agro-ecological zones in Chile. Isoenzyme studies have shown a narrow genetic distance between different strains of Chilean germplasm. This present work characterizes Chilean cultivars and regional accessions and compares them with those in Canada using the cytotaxonomic tools of chromosome formula, chromosomal asymmetry index, total chromosome length (TLC), and a detailed architectural examination of each chromosome. We found chromosome stability among all lentils as 4m+3sm. Results showed a low TLC but higher inter-chromosomal asymmetry in the accessions from Hualqui (Biobío Region) (CVCL = 31.25), a unique secondary constriction from Yumbel (Ñuble Region), and a higher TLC in Collanco (Biobío Region). Even though there was no evident karyotypic separation between Chilean and Canadian lentils, there was greater heterogeneity in TLC among cultivars in Chile than among those from Canada. It is unknown what physiological differences these cytological variants might possess. Because L. culinaris in Chile is of agronomic importance, studies of conservation are recommended to safeguard cytotaxonomic races as a national genetic resource.