The Vaccinia virus (VACV) played a pivotal role in the discovery of the 5' mRNA. Over the past decades, VACV investigation has contributed extensively to understanding the mechanism behind 5' mRNA cap synthesis. In this study, we conducted a comprehensive analysis of VACV transcripts at the level of individual mRNA molecules. Surprisingly, our findings reveal that postreplicative VACV mRNAs, which incorporate nontemplated 5' poly(A) leaders, lack the conventional 5' cap structure in vivo.