Objectives: Glial fibrillary acidic protein (GFAP) and ubiquitin Cterminal hydrolase-L1 (UCH-L1) have been studied as potential biomarkers of mild traumatic brain injury (mTBI). We report the levels of GFAP and UCH-L1 in patients with acute orthopedic injuries without central nervous system involvement and relate them to the levels in patients with CT-negative mTBI. Methods: Serum UCH-L1 and GFAP were measured from 73 patients with acute orthopedic injury. The injury types were recorded and most of the patients underwent also a head MRI. The results were compared to those found in patients with CT-negative mTBI (n = 93). Results: The levels of GFAP were higher in patients with acute orthopedic trauma than in patients with CT-negative mTBI (p = 0.026). The levels of UCH-L1 were not significantly different between these two groups. Those patients with orthopedic trauma, who had levels of either or both of these biomarkers in the upper quartile, had significantly higher levels of both biomarkers than patients with CT-negative mTBI (p < 0.001). Conclusions: Levels of GFAP and UCH-L1 were not able to distinguish patients with CT negative mTBI from patients with orthopedic trauma. Patients with orthopedic trauma and high levels of UCH-L1 or GFAP values may be falsely diagnosed as having a concomitant mTBI. This casts a significant doubt on their diagnostic value in cases with mTBI. Keywords: GFAP, UCH-L1, orthopedic injury