Trans-fatty acids are contained in partially hydrogenated vegetable oil (PHVO) and ruminant fat. Epidemiological studies have reported that a high intake of industrial trans-fatty acids such as PHVO associates with increasing risk of cardiovascular disease because of their influence on lipoproteins in plasma. In contrast, such a relationship has not been established for ruminant trans-fatty acids. These contrary health effects may be caused by the difference in the distributions of trans-octadecenoic acid (C18:1) positional isomers in PHVO and ruminant fat. However, the differences in the metabolism of trans-C18:1 positional isomers are still under debate. Therefore, many researchers have so far developed analytical methods for the separation and quantification of trans-fatty acid positional isomers by gas chromatography to reveal their metabolic character. This paper reviews the current situation of trans-fatty acid issue and the recent advances in analysis of trans-fatty acid positional isomers.