Breast milk may contain industrially produced trans fatty acids (TFAs), which can affect the content of essential fatty acids (EFAs). This could have significant implications for the child's development. The fatty acids present in breast milk can be modified by adjusting the mother's diet. The objective of this study was to determine the content of industrially produced TFAs present in colostrum, transitional milk, and mature milk produced by mothers between 18 and 45 years of age in the state of Querétaro, Mexico, based on a longitudinal observational study. The TFA content in the breast milk of 33 lactating women was analyzed using gas chromatography. The mothers’ consumption of TFAs was also estimated by analyzing a log prepared through 24‐hr dietary recall (24HR) obtained in each period. The TFA content in the mothers’ diet was similar across the colostrum, transitional milk, and mature milk phases: 1.64 ± 1.25 g, 1.39 ± 1.01, and 1.66 ± 1.13 g, respectively. The total TFA content was 1.529% ± 1.648% for colostrum; 0.748% ± 1.033% for transitional milk and 0.945% ± 1.368% for mature milk. Elaidic acid was the TFA in the highest concentration in all three types of milk. No correlation was found between the content of industrially produced TFAs in breast milk and the anthropometric measurements of the mother or between the estimated consumption of TFAs and the content of TFAs in breast milk. Elaidic acid and total content of TFAs were negatively correlated ( p < .05) with the content of docosahexaenoic acid (DHA) (0.394 ± 0.247) ( R = −0.382) in colostrum. The concentration of TFAs was found to correlate with the composition of EFAs in milk.