PURPOSE To evaluate the regenerative effect of the additional suture line when using either isografts (ISOs) or acellular nerve allografts (ANAs) placed end-to-end to span a short gap in a rat model. METHODS Rat sciatic nerves were transected and repaired with 2-cm nerve grafts (ISO or ANA). The grafts were 2 cm in length or a 1-cm segment was connected end-to-end to a 1-cm segment to yield a 2-cm length. At 8 weeks, extensor digitorum longus (EDL) muscle force and mass were measured. Nerves were harvested for histomorphometry. In a separate parallel study, the nerves were harvested 2 weeks following graft implantation to assess gene expression changes. RESULTS All grafts demonstrated regeneration across the 2-cm segment(s). The additional suture line did not result in statistical differences in the number of myelinated nerve fibers that reached the distal nerve. However, when the graft types were compared, there was a significant decrease in nerve fibers in the ANA groups. The EDL muscle mass was significantly greater by using nerve ISOs compared with ANAs, regardless of an additional suture line, but there were no statistical differences noted in EDL muscle force. Gene expression analysis did not differ owing to an additional suture line. CONCLUSIONS Minimal axonal loss and no functional deficits were identified with an additional suture line in this rodent short nerve gap model. CLINICAL RELEVANCE Placing nerve grafts in series is a viable option for treating short nerve gaps; however, the use of autografts remains preferable over the use of ANAs.