In vitro culture of sand pear (Pyrus pyrifolia) cv. Huanghua (HH) infected with apple stem grooving virus (ASGV) was conducted and the titer and variant mutations of the virus in sub-culture were analyzed. The results showed that ASGV could replicate stably in 20 successive sub-cultures. The 500 bp PCR products of ASGV in each sub-culture were cloned and used for single-strand conformation polymorphism analysis. The results demonstrated that there was a dominant molecular variant of ASGV in vitro and that the variant structure of ASGV is stable in sub-culturing. The rate of the main haplotype in the 20 passages was from 78.0% to 95.7%. To clarify the origin of subordinate haplotypes, the error rate of our RT-PCR assay (5.4× 10−4/nt) was computed and the results indicated that the mutations which appeared in sub-passages were acquired from artificial operation and not produced by viral replication.