To study the SSR loci information and develop molecular markers, a total of 435,858 unigenes in transcriptome of Polygonatum sibiricum were used to explore SSR. The distribution frequency of SSR and the basic characteristics of repeat motifs were analyzed using MISA software, and SSR primers were designed by Primer 3.0 software and then validated by PCR. Moreover, the gene function analysis of SSR Unigene was obtained by Blast. The results showed that 112,728 SSR loci were found in the transcriptome of Polygonatum sibiricum, which distributed in 435,858 unigenes with a distribution frequency of 25.86%. Mo-nucleotide and Di-nucleotide repeat were the main types, accounted for 83.83% of all SSRs. The repeat motifs of A/T and AC/GT were the predominant repeat types of Mo-nucleotide and Di-nucleotide, respectively. A total of 113,305 pairs of SSR primers with the potential to produce polymorphism were designed for maker development. One hundred and fifty-four of the 500 randomly selected primers not only produced fragments with expected molecular size but also had high polymorphism, which could accurately separate the tested varieties. The gene function of unigenes containing SSR was mostly related to the molecular function of Polygonatum sibiricum. The SSR markers in transcriptome of Polygonatum sibiricum show rich type, strong specificity, and high potential of polymorphism, which will benefit the candidate gene mining and marker-assisted breeding. The developed markers can also provide technical methods for molecular identification of intraspecific species of Polygonatum Mill. and maker-assisted breeding of superior varieties of Polygonatum Mill.