Retinoic acid inducible gene I (RIG-I) is a pattern recognition receptor (PRR) responsible for detection of nucleic acids from pathogens in the cytoplasm of infected cells and induction of type I interferon (IFN). RIG-I -specific pathogen associated molecular patterns (PAMPs) are characterized by RNA molecules with a 5'-triphosphate (5'-ppp) group and partial double-stranded composition. Although many RNA molecules capable of activating RIG-I have been described, the exact nature of viral RNAs which are responsible for triggering RIG-I activity during the course of an infection has not been extensively explored and the specificity of RIG-I for various viral RNA molecules remains largely unknown. By examining endogenous RIG-I/RNA complexes in influenza virus and Sendai virus infected cells we were able to identify viral RNA molecules which specifically associated with RIG-I during infection. We showed that in Sendai virus infected cells, RIG-I specifically and preferentially associated with the copy-back defective interfering (DI) particle RNA and not with the full-length Sendai virus genome or Sendai virus encoded mRNAs. In influenza virus infected cells RIG-I also preferentially associated with DI RNAs as well as with the shorter genomic segments.