Abstract: To explore large-scale and use the type I of microsatellite markers, microsatellite sequences in Miichthys miiuy genome transcription were analyzed and selected of polymorphism by establishing full homogenization cDNA library and random sequencing. Program of Tandem Repeat Finder (TRF) was used to select the sequences contained simple sequence repeats (SSR) from the expressed sequence tags (ESTs) of M. miiuy. Then some characterizations, such as the repeat type, time and number, the percentage of SSR and ESTs and total length, were added up based on the motif. Totally, 382 sequences contained not less than 14 bp simple repeats were obtained from 4609 ESTs of M. miiuy in our study, about 8.29% in the total database and on average about 318 bp length of EST contained a repeat sequence not less than 14 bp. All of the SSR selected were low-copies and the repeat types were abundant, including dinucleotide, trinucleotide, four-nucleotide, five-nucleotide and six-nucleotide repeat. Among the kinds of microsatellite repeats, these types, dinucleotide and trinucleotide repeat were the most abundant and it was 37.43% and 32.98% of the total microsatellite repeats, respectively. These types of microsatellites were 70.41% of the total, but the advantage motif type of each were different. The AC repeat was the most common type (75.4%) in dinucleotide repeats, while in trinucleotide repeats, the A and G combination motifs (AGG and AAG) was the most common, which was 44.75% of all motifs. At the same time, the initiative polymorphism was tested to examine the value of EST-SSR loci selected in our study. Finally, 33 pairs of primers were successfully amplified of 45 pairs of designed primers, and nine loci were polymorphism. The polymorphism loci were about 20% of the total primers. So the type of microsatellite we selected was great abundant and useful for the further study. The results provided the basis for comparing the frequency and abundance of the microsatellites distribution between species and the development of type I microsatellite markers in M. miiuy and some allied species. The nine polymorphism loci could be used in genetic diversity analysis, genome mapping and molecular assisted breeding in M. miiuy.