It is of great significance to study the human glycogen storage disease type V (GSDⅤ) caused by PYGM mutation through constructing zebrafish mutants of muscle glycogen phosphorylase gene pygma and pygmb.Two homozygous mutants were successfully obtained using CRISPR/Cas9 genome editing technique. The periodic acid Schiff staining experiment showed that there was obvious glycogen storage in the muscle and heart of pygma^-/- and pygmb^-/- compared with those of wild type (WT).The expression analysis of pygma and pygmb genes in WT at 15 early developmental stages and 12 adult tissues demonstrated that:the expression pattern of pygma and pygmb was similar at early stages, the expression of pygma and pygmb showed an overall upward trend and the increase of pygmb was greater than that of pygma from 24 hpf to 125 hpf.In the 12 tissues tested, pygma and pygmb were expressed only in a few tissues. pygma was mostly expressed in muscle tissue, followed by heart and skin; pygmb was highly expressed in heart, brain and eyes, and also expressed in muscle but at relatively lower level. Zebrafish pygma^-/- and pygmb^-/- mutants have the typical characters of GSDV with glycogen accumulation in muscle. The establishment of the zebrafish disease model provides a basis for further research on the pathogenesis, mechanism and drug screening for treatment of glycogen storage disease.