In order to solve the two-species Small Phylogeny Problem (SPP) in the duplication-loss model, a simulated annealing algorithm named SA2SP was devised for the duplication-loss alignment problem. An alignment algorithm was introduced to construct the initial solution; a labeling algorithm was used to construct the object function and obtain the evolution cost; and three intelligent neighborhood functions were introduced to generate neighborhood solutions by using the evolutionary characteristics of gene sequences. The ribosomal RiboNucleic Acid (rRNA) and transfer Ribonucleic Acid (tRNA) of four real bacterium were used to test the performance of SA2SP and Pseudo-Boolean Linear Programming (PBLP) algorithm. The experimental results show that the SA2SP algorithm has smaller evolution cost, and it is an effective method for solving the two-species SPP in the duplication-loss model.