Ultra-High Resolution LC/mass spectrometry (LC-UHRMS; Thermo Fisher Q-Exactive) was used to identify two novel isomers of hydroxylated bis(2-ethylhexyl)-tetrabromophthalate (OH-TBPH) which were unexpectedly observed in a commercial standard of TBPH. By combining ultra-high resolution (UHR) mass spectra (MS(1)), mass errors to theoretical [TBPH-Br+O](-) were 2.1 and 1.0 ppm for the two isomers, UHR-MS(2) spectra and NMR analysis; the structures of the two compounds were identified as hydroxylated TBPH with a hydroxyl group on the aromatic ring. Relatively great proportions of the two isomers of OH-TBPH were detected in two technical products, Firemaster 550 (FM-550; 0.1% and 6.2%, respectively) and Firemaster BZ 54 (BZ-54; 0.1% and 7.9%), compared to a commercial standard (0.4% and 0.9%). To simultaneously analyze OH-TBPH isomers and TBPH in samples of dust, a method based on LC-UHRMS was developed to quantify the two compounds, using negative and positive ion modes, respectively. The instrumental limit of detection for TBPH was 0.01 μg/L, which was 200-300 times better than traditional methods (2.5 μg/L) based on gas chromatography-mass spectrometry. The analytical method combined with a Florisil cleanup was successfully applied to analyze TBPH and OH-TBPH in 23 indoor dust samples from Saskatoon, Saskatchewan, Canada. Two OH-TBPH isomers, OH-TBPH1 and OH-TBPH2, were detected in 52% and 91% of dust samples, respectively. Concentrations of OH-TBPH2 (0.35 ± 1.0 ng/g) were 10-fold greater than those of OH-TBPH1 (0.04 ± 0.88 ng/g) in dust, which was similar to profiles in FM-550 and BZ-54. TBPH was also detected in 100% of dust samples with a mean concentration of 733 ± 0.87 ng/g. A significant (p < 0.001) log-linear relationship was observed between TBPH and OH-TBPH isomers, further supporting the hypothesis of a common source of emission. Relatively small proportions of OH-TBPH isomers were detected in dust (0.01% ± 0.67 OH-TBPH1 and 0.1% ± 0.60 OH-TBPH2), which were significantly less than those in technical products (p < 0.001). This result indicated different environmental behaviors of OH-TBPH and TBPH. Detection of isomers of OH-TBPH is important, since compounds with phenolic groups have often shown relatively greater toxicities than nonhydroxylated analogues. Further study is warranted to clarify the environmental behaviors and potential toxicities of OH-TBPH isomers.