Deep sequencing of small RNAs (sRNA-seq) is now the gold standard for small RNA profiling and discovery. Biases in sRNA-seq have been reported, but their etiology remains unidentified. Through a comprehensive series of sRNA-seq experiments, we establish that the predominant cause of the bias is the RNA ligases. We further demonstrate that RNA ligases have strong sequence-specific biases which distort the small RNA profiles considerably. We have devised a pooled adapter strategy to overcome this bias, and validated the method through data derived from microarray and qPCR. In light of our findings, published small RNA profiles, as well as barcoding strategies using adapter-end modifications, may need to be revisited. Importantly, by providing a wide spectrum of substrate for the ligase, the pooled-adapter strategy developed here provides a means to overcome issues of bias, and generate more accurate small RNA profiles.