EST-SSR markers were developed based on the transcriptome of Pyropia haitanensis and DNA fingerprinting was constructed using polymorphic SSR markers for the germplasm identification of the new strain SW-81 of Pyropia haitanensis. Experimental results:9 033 microsatellite loci were detected in the transcriptome data, of which the trinucleotide repeat motif was the absolute dominant type, accounting for 95%; Designing primers for microsatellite loci by Primer 3.0 software, and 187 EST-SSR primers were synthesized randomly to analyze the effectiveness, and the 92 pairs of effective primers were finally obtained, of which 42 pairs were polymorphism EST-SSR markers, with the polymorphism amplification rate 22.46%; DNA fingerprints were constructed using the polymorphism EST-SSR marker, which was suitable for 5 strains of P. haitanensis, providing an effective molecular detection method for the germplasm identification of the new strain SW-81. The results above showed that there were abundant microsatellite loci in the genome of P. haitanensis, and the gene sequence of the same loci varied among different strains, showing the polymorphism of amplification products. The polymorphism analysis based on EST-SSR marker can provide an effective means of molecular detection for the management and identification of germplasm resources.