Pepper mild mottle virus (PMMoV) causes mosaic symptoms and malformation on both leaf and fruit of pepper, reduces considerable economical yields and poses threats to human health. In this study, infectious clone of PMMoV Huludao (HLD) isolate (pCB-PMMoV-HLD) was constructed and its infectious ablility in Nicotiana benthamiana was confirmed by virions observation and Northern blot analysis. The mutant PMMoV (HLD-fsCP) that cannot express coat protein (CP) showed reduced viral accumulation but can systemically infect N. benthamiana. We constructed several chimeric mutant viruses (ZA-HB-HC, HA-ZB-HC, HA-HB-ZC and HA-ZB-ZC) by sequences substitution between PMMoV-HLD and PMMoV Zhejiang isolates (PMMoV-ZJ) and analyzed their infectious abilities in N. benthamiana and Capsicum annuum. The results showed that the chimera virus expressed by pCB-ZA-HB-HC, pCB-HA-HB-ZC and pCB-HA-ZB-ZC, but not by pCB-HA-ZB-HC, exhibited reduced infectious ability compared with wild-type PMMoV-ZJ and PMMoV-HLD, which indicated that RNA sequences required for efficient infection of PMMoV differ between the two virus isolates. The differential requirement of viral RNA sequences for efficient PMMoV infection provided theoretical value to further understand the infection and pathogenesis of PMMoV.