The genomic RNA instability of Coxsackievirus B (CVB) occurs in the infected cells. The sequence characteristics and generating mechanism of these viral RNA fragments remain unknown. In this study, the genomic segments of CVB group B type 3 (CVB3) in infected cells were amplified, cloned with 5′ rapid amplification of cDNA ends (5′ RACE) assay and sequenced. The secondary structures of their 5′ ends were analyzed. The results showed that twenty fragments derived from CVB3 genome were obtained with lengths ranging from 2 067 to 5 547 bp. The 5′ ends of these fragments mainly located in the 2Apro- and 2C-coding regions. RNA folding analysis showed that the majority of these fragments could form secondary stem-loop structure in their 5′ ends. This study indicates that incomplete RNA fragments derived from CVB genome were abundant in the infected cells. These fragments could form a partial dsRNA in their 5′ ends. Our data suggests that these fragments are not generated randomly but more likely by the cleavage of RNA endonuclease. This observation is helpful to understand the mechanism for the genome instability of CVB.