It has been reported that mammalian amelogenin exhibits heterogeneity in tooth organs with different isoforms playing different roles in enamel formation. The heterogeneity of amelogenin is reported to be generated either from alternative MK-1775 splicing of amelogenin or posttranslational processing of amelogenin protein. Mammalian amelogenin and its alternative splicing forms have been characterized, splicing forms of amelogenin gene and their functions in non-mammalian species remain largely unknown. Enamel microstructure between mammalian and non-mammalian vertebrates is significantly different in that mammalian enamel microstructure contains a prismatic, long, and parallel crystallite, while the non-mammalian vertebrate enamel contains prismless, long, and parallel crystallites. One of the mechanisms has been suggested that the complex microstructure observed in many mammals may require a range of amelogenin proteins to yield a variety of HAP crystallites in size and orientation, while the simple enamel microstructure in non-mammalian may require fewer and simpler proteins. To explore the mechanism underlying the enamel microstructure difference among mammalian and nonmammalian vertebrates, and amelogenin evolution, we previously cloned a full-length amelogenin gene and characterized the enamel formation in tooth organs from green iguana. In this study, we have demonstrated the heterogeneous amelogenin expression in the black spiny-tailed iguana tooth organs using western blot and immunohistochemistry, discovered for the first time the alternative splicing of amelogenin gene in non-mammalian vertebrate by employing RT-PCR and sequencing analysis. The results in this study revealed one predominant band with the molecular weight about 20 kDa, and two minor bands with molecular weight about 21 kDa and 8 kDa, respectively, indicating the heterogeneous amelogenin expression in iguana tooth organs. Exon X peptide in tooth organs further confirms our finding of amelogenin heterogeneous expression in the black spiny-tailed iguana. As a major tooth enamel matrix protein, amelogenin protein has been demonstrated to play a crucial role in the enamel formation and functions as a signaling molecule during tooth biomineralization. The discovery of two different splicing forms of amelogenin gene in the black spiny-tailed iguana suggest that amelogenin alternative splicing, as one of the mechanisms of amelogenin heterogeneity in tooth organs, is repeatedly employed from non-mammalian to mammalian vertebrates. Heterogeneous amelogenin protein expression in mammalian tooth organ is believed to be the result from either alternative splicing or posttranslational proteolysis of amelogenin. In this study, we employed PCR and sequence analysis to discover two amelogenin gene splicing forms in the black spiny-tailed iguana teeth. Sequencing provides definitive evidence showing that C. similis-T2S and C. similis-T2L are different splicing forms of transcripts expressed in iguana tooth organ. These findings are congruent with amelogenin protein expression pattern detected in tooth organs. Amelogenin, the major enamel matrix protein of the developing tooth organ, is highly conserved throughout most species studied. Amelogenin gene structure is similar, normally consisting of 7 exons including exon 1, 2, 3, 4, 5, 6, and 7 in which exon 4 was usually skipped during the processing of amelogenin pre-mRNA.