Markers are individual nucleotides that indicate a particular gene; the importance of selection, trait mapping, and region mapping is known. Sugarcane is a complex hybrid interspecific cross with polyploidy in nature. Five to 16 different species of the Saccharum genus have been used for sugarcane breeding, due to which sugarcane has a complex genome structure and genetic background. Genome analysis is important for genomic selection, and analyzing single nucleotide polymorphism (SNP) markers in breeding chromosome segment substitution lines (CSSLs) is important for selecting desirable traits. Marker-assisted selection is an efficient tool for selection as it focuses on screening quantitative trait loci (QTLs) and mapping the traits affecting the scenario. Genotyping of markers is done for QTL mapping, which identifies markers, such as SSRs, SNPs, etc. After genotyping and identification of markers, breeding candidates are selected, and further phenotyping is done for favorable genotypes. Favorable genotypes are tested for progeny vigor and are further analyzed for year-wise sugar recovery. The beneficial sugarcane traits are introduced in a new elite variety through molecular characterization techniques such as SSR and SNP-based markers since they are easy to design and more efficient. Generating new cultivars involves the application of biotechnology, which consists of the induction of mutations, which is done by chemical mutagens and depends on stress mechanisms. This review focuses on using and misusing various markers, their applications, challenges, and ethical considerations in sugarcane breeding programs.