The degradation of triglycerides in oil palm fruit due to an endogenous lipase in the pulp is the main reason for acidification of palm oil, which causes major economic losses and is currently mainly associated with the FLL1 gene. We designed this study to identify all the major genes controlling differences in acidity and lipase activity in the oil palm fruit mesocarp and determine a molecular markers kit to allow marker-assisted selection of commercial varieties with low acidity. Not only one gene (FLL1) but three closely linked genes including FLL1 were found and characterized in LM2T_EgCIR184O12c, a bacterial artificial chromosome sequence of 231 kb. Intra-gene PCR-based markers were designed for these genes. A QTL gene co-localization analysis for oil acidity (percentage of fatty acids released) was performed on two mapping populations. It evidenced a single major QTL at our lipase gene loci, explaining 84 to 92% of phenotypic variation, and validating the main genetic control of palm oil acidification by FLL1 and/or by the two new lipase genes. The three lipase genes had high homology to demonstrated triacylglycerol lipases. While FLL1 shows the highest expression levels, the two other genes may also contribute to oil acidity. Our molecular markers of lipase genes and the associated major QTL is an important step towards marker-assisted selection of commercial varieties with low acidity.