Background: Mild traumatic brain injury (mTBI) may rapidly alter neurocognitive function and has been associated with an increased risk for post-traumatic stress disorder (PTSD). However, neuroimaging investigations during the acute post-trauma phase remain sparse. We previously reported greater left anterior insular cortex (aIC) activation during the cognitive appraisal of fearful versus neutral emotional faces in survivors who did and did not exhibit probable PTSD at 3 months, but not at 2 weeks, after a motor vehicle collision (MVC). Given previous studies suggesting that mTBI may lead to increased cortical activation in the early post-trauma period, we hypothesize that aIC activation within 2 weeks post-MVC may be elevated among probable PTSD survivors who sustained an mTBI compared to those who did not sustain mTBI. Methods: In this secondary hypothesis-driven analysis, previously reported task-related activation at both 2 weeks and 3 months after trauma was extracted from the aIC region of interest and compared among groups of survivors diagnosed with probable PTSD at 3 months with (n = 5) and without (n = 11) mTBI in the emergency department. An exploratory analysis of change over time included two time points and additional groups of survivors without probable PTSD with (n = 10) and without (n = 12) mTBI. Results: At 2 weeks, aIC activation was significantly greater in the probable PTSD with mTBI group than in the probable PTSD without mTBI group (mean = 0.125, SD = 0.053 vs. 0.014, SD = 0.111) (Welch’s t (13.86) = 2.71, two-sided p = 0.017). aIC activation in probable PTSD survivors with and without mTBI was not significantly different at 3 months after MVC. The time and time × group interactions were not significant in brain activation and post-traumatic stress symptoms, but within-group tests suggest that decreases in post-traumatic stress symptoms over time were significant in groups without mTBI. Conclusions: These preliminary results are consistent with the hypothesized association between mTBI and greater aIC activation to negative emotional stimuli in the early post-trauma period among survivors with probable PTSD, but do not support post-trauma brain changes in survivors with mTBI, probable PTSD, both conditions, or neither condition. These preliminary findings warrant further investigation into the relationship between mTBI and probable PTSD-related cortical alterations during the acute post-trauma period.