Repetitive DNA accounts for a significant part of the eukaryotic genome, forming large clusters or being relatively uniformly distributed through euchromatic chromosome regions. Repeats make it difficult to analyze and to identify chromosomal material by its DNA composition via fluorescence in situ hybridization (FISH). In most cases, signals from unique sequences are required for identifying the chromosome regions by FISH. The review considers the feasibility, advantages, and disadvantages of conventional methods to suppress repetitive DNA hybridization, methods to construct repeat-free probes, and methods to estimate the signal intensity for chromosome-specific DNA sequences via image processing in the case of multicolor FISH. The efficiency of different techniques used to generate the DNA probes, different FISH protocols, and the processing of microscopic images depends on the genome size and structure in the species under study. Hence, several approaches to the analysis are discussed for species with extremely large genomes, rare species, and species where genomic and Cot-1 DNAs are difficult to obtain in the amounts required for suppression of repetitive DNA hybridization.