Objective This study used finite element analysis (FEA) to assess the von Mises stresses of a mandibular first premolar after removing a separated instrument with an ultrasonic technique. Methods FEA models of the original and treated mandibular first premolar were reconstructed, and three models (the original canal, size 30/taper 0.04 canal, and separated instrument removal canal) were created. Two-direction (vertical and lateral) loading patterns were simulated with a 175-N force. The maximum von Mises stresses of the models within the roots from the apex to the cervical region were collected and summarized. Results Under vertical and lateral loads, all maximal values in the three models were localized in the straight-line access region. Compared with the original model (model 1), the treated models (models 2 and 3) had greater maximum stress values from the apex to the cervical region. Greater differences in the maximum von Mises stresses between models 2 and 3 were present in the straight-line access region. Conclusions Separated instrument removal caused changes in stress distribution and increases in stress concentration in the straight-line access region of roots.