CSSLs are a complete library of introgression lines with chromosomal segments of usually a distant genotype in an adapted background and are valuable genetic resources for basic and applied research on improvement of complex traits. Chromosome segment substitution lines (CSSLs) are genetic stocks representing the complete genome of any genotype in the background of a cultivar as overlapping segments. Ideally, each CSSL has a single chromosome segment from the donor with a maximum recurrent parent genome recovered in the background. CSSL development program requires population-wide backcross breeding and genome-wide marker-assisted selection followed by selfing. Each line in a CSSL library has a specific marker-defined large donor segment. CSSLs are evaluated for any target phenotype to identify lines significantly different from the parental line. These CSSLs are then used to map quantitative trait loci (QTLs) or causal genes. CSSLs are valuable prebreeding tools for broadening the genetic base of existing cultivars and harnessing the genetic diversity from the wild- and distant-related species. These are resources for genetic map construction, mapping QTLs, genes or gene interactions and their functional analysis for crop improvement. In the last two decades, the utility of CSSLs in identification of novel genomic regions and QTL hot spots influencing a wide range of traits has been well demonstrated in food and commercial crops. This review presents an overview of how CSSLs are developed, their status in major crops and their use in genomic studies and gene discovery.