Pot-pollen (PP) is produced from the fermentation of flower pollen by stingless bees. This study aimed to analyze the fatty acid (FA) profile of Melipona seminigra pot-pollen. The samples were collected in four localities in Amazonas state, Brazil: Manaus Urban Zone (MUZ), Manaus Rural Zone (MRZ), Iranduba (IRA) and Boa Vista do Ramos (BVR). PP oil was extracted through cold extraction, and solvent evaporation was conducted under nitrogen flow. After esterification, fatty acid methyl esters (FAMEs) were separated by gas chromatography on an Agilent 68650 Series GC System (Agilent Co. Ltd, USA) equipped with a flame ionization detector and DB-23 capillary column. Analysis was performed with helium as a carrier gas, and the flow rate was 1 mL/min. FAMEs were identified by comparing the retention times in the samples to the peaks of the reference standards. The quantification was calculated using area normalization. The palmitic acid was the major saturated fatty acid (SFA) in all samples analyzed in the current study making up 21.63–22.86% of total FA. The BVR sample presented the highest monounsaturated fatty acid (MUFA) value (26.23%). Oleic acid was the main MUFA in all samples, ranging from 10.12% to 24.37% of total FA. All samples showed high contents of polyunsaturated fatty acids (PUFA), ranging from 44.94% to 59.57% of total FA. Linoleic and linolenic acids were the main constituents of PUFA in our study, contributing 21.20–40.54% and 16.26–23.50% of total FA, respectively. Among the numerous benefits attributed to these FAs are the cardioprotective, neuroprotective, and anti-inflammatory effects. The best PUFA/SFA ratio was observed in the MRZ sample (2.08). A diet with high PUFA/SFA ratios is recommended for the prevention of coronary heart diseases. Overall, in the FA profiles of most PP samples, PUFAs dominated, followed by SFA and MUFA.