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Nile tilapia is a freshwater fish of African origin, with productive and economic characteristics affecting global fish markets. The Oreochromis niloticus reference genome has a diploid set of 22 linkage groups (LGs) in addition to ungrouped sequences. A total of 42,622 genes have been identified, but 550 genomic gaps may include more. Our study focused on some of these genomic gaps, where appropriate primers were designed and then multiplied through the polymerase chain reaction (PCR) technique. From local samples, DNA was extracted and amplified with the new primers. Successful amplicons were sequenced and annotated using available bioinformatics tools. Five assessed sequences were annotated, of which three were newly predicted in O. niloticus, as mucin 1-like, and KLR genes, and SATB region. The other two sequences belonged to grid2 and trpm7 which were previously predicted. Although individual attempts to identify genomic gaps are not feasible in filling the large lack of information on the Nile tilapia genome, a good number and valuable new information has been reached. However, the following plan showed many technical problems, more time, effort and cost that could be avoided by suggesting the application of another technique, such as whole-genome sequencing, mapping, and assembly.