Abstract: To achieve swift and precise variety identification of Hippophae rhamnoides, a set of EST-SSR (express sequence tags from simple sequence repeat) markers was developed. These markers exhibit high polymorphism, stability and generality, and have allowed the construction of the fingerprint of H. rhamnoides. For the transcriptome sequencing data, the young and fresh leaves of 'Shiyou 1' were selected for RNA extraction and cDNA library construction. Additionally, young and healthy leaves from 42 varieties of H. rhamnoides were collected. The EST-SSR markers were designed by using microsatellite identification tool (MISA) and Primer 3 (version 2.3.4). PCR and TP-M13-SSR combined capillary electrophoresis examined fluorescence signals and peak emergence for 42 varieties. Amplified product lengths and genetic information of EST-SSR markers were assessed by using GeneMarker (version 2.2.0), Popgene (version 1.32), Cervus (version 3.0.7), and Convert (version 1.31). Genetic relationship analysis of the varieties was conducted by using NTSYSpc (version 2.10e). As a result, a total of 6 196 SSR loci were obtained, with 182 motifs types among all the SSR loci. The dominant motif was (A/T) n. The lengths of the SSR motifs were 10-21 bp, accounting for 81.58% of all SSR sequences. The main types of SSR repeat motifs were mono-nucleotide (48.72%), di-nucleotide (22.68%) and tri-nucleotide (18.85%). 28 pairs of primers were developed and a total of 193 allele loci in the 42 varieties were detected. The genetic diversity indexes, including the mean number of observed alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information content (PIC) and Shannon information index (I) were 6.964, 3.495, 0.617, 0.671, 0.623 and 1.384, respectively. The genetic similarity coefficients between varieties ranged from 0.601 to 0.990, as determined by UPGMA clustering. At a coefficient of 0.694, varieties formed two groups, while at coefficient to be approximately 0.740 2, they grouped into three. Using six selected primers, the fingerprint of H. rhamnoides was constructed for rapid and accurate variety identification. This study provides the theoretical basis and molecular-level data support for variety identification, fingerprint construction, genetic diversity and relationship analysis of H. rhamnoides.