Abstract Introduction Throughout history, thousands of medicinal and aromatic plants have been widely utilised by people worldwide. Owing to them possessing of valuable compounds with little side effects in comparison with chemical drugs, herbs have been of interest to humans for a number of purposes. Diosgenin, driven from fenugreek, Trigonella foenum‐graecum L., has extensively drawn scientist's attention owing to having curable properties and being a precursor of steroid hormones synthesis. Nonetheless, complete knowledge about the biosynthesis pathway of this metabolite is still elusive. Objective In the present research, we isolated the full‐length CDS of 14 genes involving in diosgenin formation and measured their expression rate in various genotypes, which had illustrated different amount of diosgenin. Methodology The genes were successfully isolated, and functional motifs were also assessed using in silico approaches. Results Moreover, combining transcript and metabolite analysis revealed that there are many genes playing the role in diosgenin formation, some of which are highly influential. Among them, ∆ 24 ‐reductase, which converts cycloartenol to cycloartanol, is the first‐committed and rate‐limiting enzyme in this pathway. Additionally, no transcripts indicating to the presence or expression of lanosterol synthase were detected, contradicting the previous hypothesis about the biosynthetic pathway of diosgenin in fenugreek. Conclusion Considering all these, therefore, we propose the most possible pathway of diosgenin. This knowledge will then pave the way toward cloning the genes as well as engineering the diosgenin biosynthesis pathway.