The aim of this study was to evaluate the biohydrogenation in dairy cows fed unsaturated fatty acid by use of reticular and omasal sampling as an alternative to sampling from the abomasal canal. Four Holstein dry cows cannulated in the rumen and abomasum (602 ± 21 kg of body weight; mean ± SD) were assigned randomly into a 4 × 4 Latin square design experiment, fed the following diets: (1) control (C), without unsaturated fatty acid supplementation; (2) soybean oil (SO), addition of 3% of refined soybean oil in DM basis of total diet; (3) whole raw soybean (WS), addition of 16% of whole raw soybeans in DM basis of total diet; and (4) calcium salts of unsaturated fatty acids (CSFA), addition of 3% of CSFA in DM basis of total diet. Nutrient flow was calculated using the reconstitution system based on 3 markers (cobalt-EDTA, ytterbium chloride and indigestible NDF). Large and small particles and the fluid phase were recovered from digesta collected of different sites. Samples were collected 8 times during 20 4 h and were combined into one pool sample for further reconstitution. Data were analyzed using PROC MIXED of SAS 9.1 using the Tukey test to test pair wise comparisons. There were not effects of sampling sites, on biohydrogenation of 18:3 (85.6; 97.9 and 93.2% of intake for to digesta reticular, omasal and abomasal respectively). However, the biohydrogenation of C18:2 (84.2; 93.4 and 85.1% of intake for to digesta reticular, omasal and abomasal respectively) (P < 0.01) and C18:1 (63.2; 83.7 and 62.9% of intake for to digesta reticular, omasal and abomasal respectively) were overestimated by use sampling omasal in relation to use reticular sampling and abomasal (P < 0.01).The reticular sampling technique provided reliable estimates for biohydrogenation of 18:2 and 18:1. The omasal sampling technique can overestimate to biohydrogenation.