Nucleotide variations that do not alter the protein-coding sequence have been routinely considered as neutral. In light of the developments we have seen over the last decade or so in the RNA processing and translational field, it would be proper when assessing these variants to ask if this change is neutral, good or bad. This question has been recently partly addressed by genome-wide in silico analysis but significantly fewer cases by laboratory experimental examples. Of particular relevance is the effect these mutations have on the pre-mRNA splicing pattern. In fact, alterations in this process may occur as a consequence of translationally silent mutations leading to the expression of novel splicing isoforms and/or loss of an existing one. This phenomenon can either generate new substrates for evolution or cause genetic disease when aberrant isoforms altering the essential protein function are produced. In this review we briefly describe the current understanding in the field and discuss emerging directions in the study of the splicing mechanism by integrating disease-causing splicing mutations and evolutionary changes.