Saturated straight- and branched-chain 3-hydroxy fatty acids (3-OH FAs) of 10–18 carbon chain lengths were determined in saliva from 27 individuals with chronic periodontitis and 18 healthy individuals by using gas chromatography–tandem mass spectrometry. Of the 14 different 3-OH FAs detected, 3-OH-C i17:0 was the most abundant in the periodontitis samples while 3-OH-C 14:0 was the most abundant in the healthy individuals. Considering the relative percentages of 3-OH-C 12:0 , 3-OH-C 14:0 , 3-OH-C i17:0 , and 3-OH-C 17:0 , 95.6% of all cases were correctly classified as healthy individuals or periodontitis patients by means of discriminant analysis. The sensitivity, specificity, positive predictive value and negative predictive value of 3-OH FA analysis in diagnosing peridontitis were, respectively, 0.92, 1.00, 1.00, and 0.90. The results indicate that 3-OH FA analysis of saliva samples is a useful diagnostic method in chronic periodontitis.