Using sequence alignment-free comparison method to study microbial flora classification is a hot topic in bioinformatics． In this paper，SeqDistK，the sequence alignment-free comparison software based on k-mer statis- tics，is presented． The open source software package can be obtained from https: ∥github． com /htczero /SeqDistK． SeqDistK has the advantages of fast calculation and high accuracy in microbial flora classification，and has the po- tential to adapt to large-scale data research． By adopting SeqDistK to cluster 63 distance matrices of 16S rＲNA gene sequences，it is found that the clustering results are basically consistent with the existing classifications，which means that SeqDistK can accurately classify microbial flora clustering samples and provides effective software for phylogenetic analysis in molecular biology．