Abstract: Objective To reconstruct tumor clonal haplotypes based on the third- generation sequencing data to effectively identify tumor heterogeneity. Methods We developed an algorithm for extracting somatic mutational event from the mixed tumor data and determining the connection weight of each somatic cell mutation site through the probability function. A reconstruction algorithm of the haplotype was designed based on the maximum spanning tree, and following the principle of inheritance between tumor clones, the connection pattern was determined at each mutation site in the clonal maximum spanning tree in a stepwise manner. The number, ratio and evolution of the sub-clones were estimated using the depth stripping method. Results In the simulation experiments, we analyzed the accuracy of the algorithm based on 4 indexes, namely the coverage, read length, subclone number and somatic variant rate, and the results demonstrated a good robustness of the algorithm. The results of the experiments showed that the mean sub-clone haplotypes accuracy exceeded 97%, suggesting that this algorithm significantly outperformed the previous methods. Conclusion The proposed method can accurately reconstruct tumor subclonal haplotypes and clarify the process of tumor clonal evolution, and can thus provide a theoretical basis for tumor heterogeneity research and assist in clinical decision-making.