Tomato or (Solanum lycopersicum) is the most prevalent vegetable all over the world; it is an important source of minerals, vitamins and amino acids. This study was designed to estimate genetic diversity in LOC101 gene that is coding for pyruvate kinase enzyme in 14 commercial tomato cultivars. Pyruvate kinase is serious enzyme of glycolytic pathway as it stimulates the final step of glycolysis. Genetic diversity was estimated using two marker systems; first pyruvate kinase start codon surrounding regions using characterized marker (SCoT5). Second; specific molecular marker for LOC101 gene.  The results showed that the genetic diversity related to Pyruvate kinase start codon using SCoT5 primer was 75%. The 14 tomato cultivars were divided into 5 clusters in relation with genetic diversity. Similarity matrix and dendrogram revealed that the highest diversity was between cultivars Advantage 2 with cultivars; Floradade and Vt 916 hybrid followed by the diversity between (Floradade and Super queen, Peto 86 and Super strain b) and Super queen with Advantage 2, Uc _97-3, Vt 916 hybrid, Edkawy and Sahrawy 1 hybrid. also  high diversity was recorded between (Vt 916 hybrid and Peto 86) and each of Advantage 2, Edkawy, Sahrawy 1 hybrid and Uc _97-3 with Super marmand, Rio pilcomojo, Safa 2033 and Radwa f1. The Amplification of LOC101 gene showed the existence of one common fragment of nearly 725 bp in the entire examined cultivars. These results present good sources of genetic diversity that can help breeders to estimate genetic diversity and relationships in substantial biological processes such as glycolysis.