Molecular sexing in wild and domestic animals has becoming an important issue in several fields including reproduction. X and Y chromosome-specific sequence differences of the amelogenin genes (AMELX and AMELY) have been described in different mammalian species and used for sex determination. We studied the possibility to use sequence variability between the porcine AMELX and AMELY genes for sex determination in pigs. Sequence analysis of about 400 bp of intron 3 of the porcine amelogenin genes showed the presence of a 9-10 bp deletion in AMELY gene compared to AMELX sequences. Moreover, one single nucleotide polymorphism (SNP) was detected for the AMELY sequence. Four other SNPs and 1 bp insertion differentiated three AMELX haplotypes indicating an unexpected quite high nucleotide diversity for a chromosome X region. Two sex determination assays targeting the 9-10 bp difference between AMELX and AMELY were developed. Assessment of the accuracy of the amelogenin assays to correctly sex individuals was tested on 329 pigs belonging to different breeds/lines. All analysed animals were correctly sexed with the new designed amelogenin tests. No amplification was obtained in human, cattle, goat, sheep, and horse genomic DNA. These assays can be used for sex diagnosis of small amounts of genomic DNA (20 pg) obtained from different sources including embryo biopsies, hair, meat, and other biological specimens. Thus, apart from the application in the reproduction field, these tests can be useful in several other sectors including forensics, archaeozoology, meat production, and processing as well as for quality control in sample identification.