We have developed a new method, employing an assembly approach, to build gene models and identify alternative isoforms. We have been using this method to study alternative splicing in chickens from two inbred lines that are resistance and susceptible to Marek’s disease (MD). The method identified many novel genes and isoforms that are not included in existing gene models. Isoforms were confirmed by reads that mapped across the exons. All kinds of alternative splicing events were detected including cassette exons, alternative splice sites, alternative 5’ and 3’ end, mutually exclusive exons and intron retention. Moreover, the method has successfully detected alternative isoforms between two chicken inbred lines that might contribute to genetic resistance to MD. Furthermore, this method does not rely on existing gene annotations, therefore, it can be applied to study alternative splicing in any organism.