This study aimed to evaluate the methylation performances of three reagents (3 N HCl-MeOH, 14% $BF_3$-MeOH, and 24 N $H_2SO_4$-MeOH) on single or mixed fatty acids. Results showed that 24 N $H_2SO_4$-MeOH (93.00%) had the best cis-9, trans-11 CLA methylation performance compared with that of 3 N HCl-MeOH (7.96%) or 14% $BF_3$-MeOH (65.49%). There were no obvious differences in the composition of major fatty acids in soybean oil or extruded full-fat linseed after methylation by these reagents. On the contrary, the individual fatty acids showed various responses to these three reagents in chicken breast meat and chicken drum stick. In milk, no obvious differences were observed except for cis-9, trans-11 CLA. These data demonstrate that the methylation performances of the three reagents varied with the kinds of sample origins to be analyzed. Therefore, prudent careful selection of the methylation reagent should be carried out before the analysis.