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Сhromosome‑length genome assemblies are the gold standard for de novo whole genome sequencing. The availability of high-quality vertebrate reference genomes led to significant advances in functional, comparative and population genomics. Problematic steps in constructing a reliable reference genome are the correct orientation of contigs and scaffolds relative to each other and the assembly of superscaffolds into structured chromosomal scaffolds, as well as the sequencing and positioning of genomic regions with repeated sequences or segmental duplications. Many of the above problems are overcome by integrating genome sequencing data with cytogenetics data. A combined approach that assembles the genome down to the chromosome level is much more successful in answering fundamental biological questions than genomic or cytogenetic approaches alone. Unfortunately, integration of genome sequencing data with cytogenetics data is often not carried out, which leads to inconsistency in chromosome numbering even for species for which standardized karyotypes are available, incorrect orientation of scaffolds, incorrect assessment of chromosome sizes due to failure to take into account heterochromatic blocks, and other problems. The purpose of this work is to draw attention to the importance of understanding and applying a combined approach when performing chromosome‑length genome assemblies. Using the example of fish, reptiles and various groups of mammals we show that integration of cytogenetic and bioinformatics data for correct chromosome-length genome assemblies is of great importance, making it possible to correct the orientation of scaffolds and to match chromosome scaffolds with chromosomes described by molecular cytogenetic methods in representatives of different vertebrate taxa.