MOTIVATION: Small interfering RNAs (siRNAs) are produced from much longer sequences of double-stranded RNA precursors through cleavage by Dicer or a Dicer-like protein. These small RNAs play a key role in genetic and epigenetic regulation; however, a full understanding of the mechanisms by which they operate depends on the characterization of the precursors from which they are derived. RESULTS: High-throughput sequencing of small RNA populations allows the locations of the double-stranded RNA precursors to be inferred. We have developed methods to analyse small RNA sequencing data from multiple biological sources, taking into account replicate information, to identify robust sets of siRNA precursors. Our methods show good performance on both a set of small RNA sequencing data in Arabidopsis thaliana and simulated datasets. AVAILABILITY: Our methods are available as the Bioconductor (www.bioconductor.org) package segmentSeq (version 1.5.6 and above).