Aim: To develop an analytic method for identifying tissue-specific (TS) genes from RNA-seq data. Materials & methods: Based on a negative binomial distribution, we develop a statistical method containing consecutive procedures incorporating data variability from replicates in each tissue. Results: Simulations show that our approach can effectively identify at least 94% of the truly TS genes if the sample size is 3 and at least 84% of the TS genes detected by our method are truly TS genes. We illustrated the utility of our method in an in-house RNA-seq project and produced sensible results. Conclusion: Our approach not only directly works on discrete data but also naturally incorporates data variability. It works effectively for detecting TS genes.