Global investigation of the 3′ extremity of mRNA (3′-terminome), despite its importance in gene regulation, has not been feasible due to technical challenges associated with homopolymeric sequences and relative paucity of mRNA. We here develop a method, TAIL-seq, to sequence the very end of mRNA molecules. TAIL-seq allows us to measure poly(A) tail length at the genomic scale. Median poly(A) length is 50–100 nt in HeLa and NIH 3T3 cells. Poly(A) length correlates with mRNA half-life, but not with translational efficiency. Surprisingly, we discover widespread uridylation and guanylation at the downstream of poly(A) tail. The U tails are generally attached to short poly(A) tails (<25 nt), while the G tails are found mainly on longer poly(A) tails (>40 nt), implicating their generic roles in mRNA stability control. TAIL-seq is a potent tool to dissect dynamic control of mRNA turnover and translational control, and to discover unforeseen features of RNA cleavage and tailing. Display omitted •We develop TAIL-seq that reveals the transcriptome-wide landscape of RNA 3′ ends•Poly(A) length correlates with mRNA half-life, but not with translation efficiency•We found widespread uridylation and guanylation at the 3′ ends of poly(A) tail•The U tails are generally found on short poly(A) tails (<25 nt) Eukaryotic mRNAs have poly(A) tails that can affect mRNA stability and translational activity. Chang et al. describe a method, called TAIL-seq, that allows high-resolution, transcriptome-wide investigation of 3′ end sequences. They use this approach to profile 3′ ends and to identify several different modifications at the ends of poly(A) tails.