Current studies on cable harness layouts have mainly focused on cable harness route planning. However, the topological structure of a cable harness is also extremely complex, and the branch structure of the cable harness can affect the route of the cable harness layout. The topological structure design of the cable harness is a key to such a layout. In this paper, a novel multi-branch cable harness layout design method is presented, which unites the probabilistic roadmap method (PRM) and the genetic algorithm. First, the engineering constraints of the cable harness layout are presented. An obstacle-based PRM used to construct non-interference and near to the surface roadmap is then described. In addition, a new genetic algorithm is proposed, and the algorithm structure of which is redesigned. In addition, the operation probability formula related to fitness is proposed to promote the efficiency of the branch structure design of the cable harness. A prototype system of a cable harness layout design was developed based on the method described in this study, and the method is applied to two scenarios to verify that a quality cable harness layout can be efficiently obtained using the proposed method. In summary, the cable harness layout design method described in this study can be used to quickly design a reasonable topological structure of a cable harness and to search for the corresponding routes of such a harness.