A 'stepwise chromosome evolution model' is proposed to elaborate the two stages of the sex chromosome evolution of Drosophila albomicans. To explain the occurrence of both a 3-X and a 3-Y in this species it is proposed that these chromosomes evolved sequentially. The selection forces acting in the two stages are different. The genic composition in the first fused sex chromosome of D. albomicans was supposed to be selectively superior to the primitive unfused chromosomes, thus the fused chromosome could overcome the meiotic structural incompatibility and be maintained in the population during the first stage. When the second fusion occurred, the newly fused sex chromosome was preferentially selected, as it could provide a meiotic pairing partner for the first fusion. The fused sex chromosomes were eventually fixed in the population. Based on this model, we tried to determine which fused chromosome, 3-X or 3-Y, established first. The karyotyping result of four experimental hybrid populations showed that the fused 3-X, as opposed to 3-Y, chromosome has higher fitness values. However, without the coexistence of 3-X, 3-Y is excluded rapidly. Therefore, 3-X is likely to be the first fused chromosome in the stepwise chromosome evolution model. The formation and fixation of the fused 3-Y chromosome occurred subsequently.