Healthy seedlings need to be transplanted into vacancy cells in tray instead of unhealthy ones. It is an important step of seedling tray cultivation in the facility agriculture. The end-effector leaved the origin, and grasped the health seedlings one by one from the transplanting tray to the aim tray. And then it was back to the origin until the all vacancy cells has been filled. This process formed the automatic transplanting path. The seedling transplanting path problem was similar to traveling salesman problem which the whole path length was objective function.A method was developed for optimization of seedling transplanting path based on genetic algorithm. The effectiveness of the algorithm was analyzed by experiment. The numerical simulated results showed that compared with the fixed sequential method, the path planned by genetic algorithm was shortened by 3.7 m for transplanting fifty seedlings. The algorithm’s average operation time was 0.65 s. The transplanting path was optimized, and operation time meets the transplanting robot’s real-time requirement. The work efficiency of transplanting was improved. 