Ultrahigh-throughput sequencing of cDNA (RNA-Seq) is an invaluable resource for investigating alternative splicing in an organism. Alternative splicing is a form of posttranscriptional regulation in which primary RNA transcripts from a single gene can be spliced in multiple ways leading to different RNA and protein products. In plants and other species, it has been shown that many genes involved in circadian regulation are alternatively spliced. As new RNA-Seq data sets become available, these data will lead to new insights into links between regulation RNA splicing and the circadian system. Analyzing RNA-Seq data sets requires software tools that can display RNA-Seq read alignments alongside gene models, enabling assessment of how treatments or developmental stages affect splicing patterns and production of novel variants. The Integrated Genome Browser (IGB) software program is a free and flexible desktop tool that enables discovery and quantification of alternative splicing. In this protocol, we use IGB and a cold-stress RNA-Seq data set to examine alternative splicing of Arabidopsis thaliana LHY, a circadian clock regulator. IGB is freely available from http://www.bioviz.org .