The feasibility of Raman spectroscopy in combination with partial least-squares (PLS) regression for the determination of individual or grouped trans-monounsaturated fatty acids (trans-MUFA) and conjugated linoleic acids (CLA) in milk fat is demonstrated using spectra obtained at two temperature conditions: room temperature and after freezing at -80 °C. The PLS results displayed capability for direct semiroutine quantification of several individual CLA (cis-9,trans-11 and trans-10,cis-12 C18:2) and trans-MUFA (trans-4-15 C18:1) in minor concentrations (below 1.0 g/100 g of milk fat). Calibration models were based on reference data cross-correlation or determined by specific scattering signals in the Raman spectra. Distinct bands for trans-MUFA (1674 cm(-1)) and CLA (1653 cm(-1)) from the trans isolated and cis,trans conjugated C ═ C bonds were identified, as well as original evidence for the temperature effect (new bands, peak shifts, and higher intensities) on the Raman spectra of fatty acid methyl ester and triacylglyceride standards, are supplied.