This study evaluated the dissolved organic carbon (DOC) composition of two surface waters in Manitoba, Canada, the Red and Assiniboine Rivers, to identify DOC fractions with the greatest potential to form trihalomethanes (THMs). The DOC was fractionated into six fractions based on hydrophobicity and acid base functionality: hydrophobic acid (HPOA), hydrophobic base (HPOB), hydrophobic neutral (HPON), hydrophilic acid (HPIA), hydrophilic base (HPIB), and hydrophilic neutral (HPIN). The trihalomethane formation potential (THMFP) of the isolated fractions was measured. Fractions collected from the Red River showed significant variation in the specific THMFP, normalized to DOC concentration, ranging from 23 to 1,372 μg THM/mg DOC, while the Assiniboine River THMFP ranged from 11 to 575 μg THM/mg DOC. Although the DOC composition of the two rivers was similar, the THMFP for the fractions differed significantly. The Red River HPOB and HPIB fractions had the highest normalized THMFP of 1,002 μg THM/mg DOC and 1,372 μg THM/mg DOC, respectively, while the HPOA fraction showed the lowest with 23 μg THM/mg DOC. Comparatively, the HPOA fraction from the Assiniboine River was found to have a higher specific THMFP of 478 μg THM/mg DOC. These findings suggest that THM formation is dependent on unique organic composition of the local water and that caution should be taken when estimating THMFP from DOC fractions isolated from different water sources.