Phosphoglycerate kinase 1 (PGK1) and pyruvate kinase M2 (PKM2) have been identified as the postmortem meat quality biomarkers. However, the precise molecular mechanism through which they affect and regulate the development of meat quality remains unclear. In this work, the high‐ and low‐activity groups (n = 10) were selected from 60 lamb muscles at 24 h postmortem based on the activity levels of PGK1 and PKM2. The metabolomic, proteomic, and transcriptomic analyses combined with deeply integrated multi‐omics analysis were used to elucidate the mechanisms by which PGK1 and PKM2 characterize meat quality. The results indicated that glycolysis played a crucial role in regulating PGK1 and PKM2 activity at the metabolome, proteome, and transcriptome levels. In glycolysis pathway, we identified several key components closely related to PGK1 and PKM2 activity, including differential metabolites (adenosine triphosphate, adenosine diphosphate, glucose‐6‐phosphate, nicotinamide adenine dinucleotide phosphate, fructose‐6‐phosphate, dihydroxyacetone phosphate, 3‐phosphoglycerate, NAD+ nicotinamide adenine dinucleotide, lactate, and pyruvate), different abundance proteins (lactate dehydrogenase B and fructose bisphosphate aldolase B), and differentially expressed genes (hexokinase and fructose‐1,6‐bisphosphatase 1). It was concluded that PGK1 and PKM2 may affect the formation of meat quality by regulating these critical substrates. Additionally, PGK1 and PKM2 could also affect the tricarboxylic acid cycle, oxidative phosphorylation, and muscle contraction in postmortem and then influence meat quality. This integrative omics study offers valuable insight into unraveling the molecular mechanisms underlying postmortem meat quality development.