Bovine milk fat (MF) is considered to be one of the most complex fats, as it can consist of 400 FA which are non‐randomly esterified into the TAG molecule. This study aims to determine specific differences on MF TAG profile between summer and winter cow's milk, and to study the feasibility of TAG fragmentation on non‐chemically hydrogenated MF using MALDI‐TOF/TOF MS with post source decay. The most notable discrimination between summer and winter MF is seen in polyunsaturated TAG, which are more abundant in summer. The feasibility of MF TAG fragmentation in non‐chemically hydrogenated TAG is assessed using bulk MF and TAG previously separated according to degree of unsaturation. This last approach shows to be the most suitable. We show that sample concentration and abundance ratio with neighboring TAG affects TAG fragmentation. Non‐chemically hydrogenated MF TAG has not been previously fragmented. Furthermore, this is the first time that the conditions affecting TAG fragmentation in MF are reported. Practical Application: TAG constitute the fundamental unit of fat crystals. Hence, fat crystal network is affected by variations in TAG profile. Understanding the variability of MF TAG profile between seasons may help to explain differences on crystallization behavior seen through the year in industrial processes. Moreover, TAG fragmentation allows determination of FA composition of individual TAG. This information is needed when studying the mechanism of FA acylation into TAG. For instance, when studying the effect of genetic variation of diacylglycerol acyl transferase isomer one in cows. The reported technique increases the capacity of MALDI‐TOF MS in lipidomic analysis. This study aims to determine specific differences in milk‐fat triacylglycerol (TAG) profiles between summer and winter cow's milk, and to study the feasibility of TAG fragmentation on non‐chemically hydrogenated milk fat using MALDI‐TOF/TOF MS with post source decay.