Chromosome structural rearrangements play a significant role in karyotype evolution and speciation. These rearrangements pose challenges for precise karyotyping, leading to asymmetric chromosomes and complicating the assembly of a genus pan-genome for crops and their wild relatives. Lens culinaris, an important cool-season legume primarily cultivated in India and Canada, is the cultivated species among six wild relatives. All seven species of Lens face significant challenges due to chromosomal rearrangements, ranging from introgression issues to difficulties in developing a precise karyotype and advancing genomic studies. Using the gene synteny analysis between the cultivated Lens species and six wild relatives, we developed cross-species oligo-FISH (Fluorescent in situ hybridization) probes aiming to further attest to the genome assembly and synteny analysis of Lens species. Roots of seven Lens spp. accessions were harvested and used for chromosome spread preparations. Those slides were then used for Oligo-FISH experiments, where the DNA present in the slides was denatured, and a set of red and green oligo probes was hybridized to the chromosomes. Pictures were taken using a fluorescence microscope. The combination of both oligo sets/probes resulted in a distinct pattern for each Lens spp. chromosome, allowing the inference of the most precise karyotype to date for six Lens species. The number of oligo probe signals reflects the species’ phylogenetic proximity, while the distribution of those signals changed drastically within the same gene pool. The karyotyping of Lens confirmed the proper assignment of chromosomes in the genome assemblies and validated the rearrangements detected in the synteny analysis. Differences in the assembly probe prediction and the oligo-FISH results were used to improve the assemblies. The results attest to a higher sequence-level similarity among the closest related species despite the occurrence of several structural changes among them. The oligo-FISH probes can be used in conjunction with plant genome assembly projects, supporting the delivery of a precise representation of their physical chromosomes.