In order to develop new wheat molecular markers,EST-SSR markers were developed based on the results of transcriptome sequencing of Guizimai No.1.The chromosome location,polymorphism of developed markers were studied.The results showed that 8 749 EST sequences containing SSR loci were obtained from 119 572 total unigenes sequences,accounting for 7.3%,with an average of 1 SSR per 8.04 kb.The distribution characteristic of SSR types in EST sequences was that the trinucleotide repeat was the most,and pentanucleotide and hexanucleotide repeats were less;A/T repeat was the most in the single nucleotide repeats,AG/CT repeat was the most in the dinucleotide repeats,and CCG/CGG repeat was the most in trinucleotide repeats. Primers were successfully designed for 5 503 EST sequences containing SSR loci,accounting for 62. 9% of the total EST sequences; 341 effective EST-SSR primer pairs were screened;341 EST-SSR primer pairs were located using 16 Chinese Spring nulli-tetrasome system materials,and 153 EST-SSR primer pairs were located in 201 loci,involving 16 chromosomes except 3A,5A,3B,4B and 1D,of which primer pairs were the most on 5B chromosome,up to 23 primer pairs; 53 polymorphic EST-SSR primer pairs were screened out, and genetic diversity analysis of 52 wheat germplasms showed that 18 EST-SSR primer pairs could distinguish 52 wheat germplasms one by one.