Palm oil adulteration in dairy products poses significant concerns for food authenticity, consumer protection, and regulatory compliance. This study presents a validated combined molecular and chromatographic detection strategy to detect and quantify palm oil adulteration in yogurt fat. Yogurt fat samples were fortified with palm olein at concentrations ranging from 1 to 100% (w/w). DNA was extracted using a modified CTAB protocol and analysed for the oil palm-specific MT3-B gene via conventional and quantitative PCR (qPCR). Concurrently, Gas Chromatography–Flame Ionization Detection (GC-FID) was used to quantify phytosterols in the unsaponifiable lipid fraction. To address this issue, we developed a dual-analytical strategy combining qPCR targeting the chloroplast MT3-B gene of oil palm (Elaeis guineensis) and gas chromatography for sterol profiling. The qPCR assay demonstrated high specificity and sensitivity, with a detection limit of 0.01 ng and a quantification limit of 0.02 ng. GC-FID analysis showed a dose-dependent increase in phytosterol content, with pronounced rises at 50% and 100% substitution. A strong correlation (r = 0.89) was observed between qPCR-detected DNA levels and GC-measured phytosterol concentrations. To evaluate real-world applicability, the method was applied to 15 commercial yogurt samples, all of which tested positive for molecular and chemical markers of palm oil. This integrated approach offers a rapid and reliable strategy for detecting palm oil adulteration, supporting food quality assurance and regulatory enforcement.

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