The frequency distribution of mutation-induced aberrant 30 splice sites (30ss) in exons and introns is more complex than for 50 splice sites, largely owing to sequence constraints upstream of intron/ exon boundaries. As a result, prediction of their localization remains a challenging task. Here, nucleotide sequences of previously reported 218 aberrant 30ss activated by disease-causing mutations in 131 human genes were compared with their authentic counterparts using currently available splice site prediction tools. Each tested algorithm distinguished authentic 30ss from cryptic sites more effectively than from de novo sites. The best discrimination between aberrant and authentic 30ss was achieved by the maximum entropy model. Almost one half of aberrant 30ss was activated by AG-creating mutations and 95% of the newly created AGs were selected in vivo. The overall nucleotide structure upstream of aberrant 30ss was characterized by higher purine content than for authentic sites, particularly in position 3, that may be compensated by more stringent requirements for positive and negative nucleotide signatures centred around position 11. A newly developed online database of aberrant 30ss will facilitate identification of splicing mutations in a gene or phenotype of interest and future optimization of splice site prediction tools.