Fatty acid composition of intramuscular, intermuscular, subcutaneous, omental and kidney knob fat depots of eighty male light lambs (±21kg live weight) from five Spanish sheep breeds was analysed. Fat depot, anatomical depot location (internal, external and intramuscular), breed (Spanish Merino, Grazalema Merino, Churra Lebrijana, Segureña and Montesina), weaning type (weaning at 45 days after birth or no weaning) and subcutaneous fat thickness factors were analyzed using a statistical model to quantify their contribution to the variation of each fatty acid. Production system was the main factor to explain variations in overall fatty acid profiles (34.68%). However, for several fatty acids and indices (arachidonic, linoleic, PUFA, n-3/n-6) anatomical depot location was the most significant factor. Feeding system explained 65.49% of CLA variance, indicating a strong influence of suckling period length on CLA deposition in lambs' fat. Moreover, due to the lack of interaction between anatomical depot location or depot and breed type or weaning system for total CLA, for future research only one depot would be enough to study the effect of those factors on CLA levels.