Non-communicable diseases are projected to become the most common causes of death in Africa by 2030. The impact on health of epidemiological and nutritional transitions in sub-Saharan Africa remains unclear. To assess the trends of dietary fatty acids over time in Uganda, we examined fatty acids in serum collected from individuals in rural south-west Uganda, at three time points over two decades. Independent cross-sectional samples of 915 adults and children were selected from the general population cohort in 1990 (n 281), 2000 (n 283) and 2008 (n 351). Serum phospholipid fatty acids were measured by GC. Multivariate regression analyses were performed to compare the geometric means of fatty acids by time period. Serum fatty acid profiling showed high proportions of SFA, cis-MUFA and industrial trans-fatty acids (iTFA), likely to be biomarkers of high consumption of palm oil and hydrogenated fats. In contrast, proportions of n-6 and n-3 PUFA from vegetable oils and fish were low. From 1990 to 2008, serum phospholipids showed increases in absolute amounts of SFA (17·3 % increase in adults and 26·4 % in children), MUFA (16·7 % increase in adults and 16·8 % in children) and n-6:n-3 PUFA (40·1 % increase in adults and 39·8 % in children). The amount of elaidic acid, iTFA from hydrogenated fats, increased in children (60·1 % increase). In this rural Ugandan population, we show evidence of unfavourable trends over time of dietary fatty acids.