Polyadenylation adds a poly(A) tail to an mRNA, which is an essential step for the choice of mRNA isoforms and the regulation of mRNA level in eukaryotes. Therefore, defects in polyadenylation profoundly alter cell viability, growth, and development. Although previous studies based on evolutionary conservation identified a few elements in the 3’ UTR, the function of other sequences is still mysterious, partly because the high level of degeneracy in coding the signal of polyadenylation in the 3’ UTR. To systematically investigate the function of 3’ UTR sequences in regulating polyadenylation, we generated a library containing 3,628 yeast strains, with a variant of 3’ UTR inserted right after a GFP coding sequence in each strain. We quantified the polyadenylation efficiency for each variant by calculating the proportion of the readthrough transcripts and correlated it with sequence properties. We identified several strategies by which polyadenylation efficiencies are coded in the 3’ UTR. Particularly, guanine-rich motifs play a central role in regulating polyadenylation efficiency. We further confirmed that sequences of 3’ UTR in yeast genome are largely in agree with the coding strategies we identified from our 3’ UTR variant library. This study expands our understanding on the coding rules and evolutionary dynamics of 3’ UTR, which together pave the road for the ultimate goal of understanding every nucleotide in a genome.