Single nucleotide variants, such as SNPs, have been extensively exploited to decode population genetic structure, phylogenetic relationships, and traits of commercial interest in aquatic species. Aquaculture research has applied high-density SNP markers to guide breeding programs based on GWAS approaches. However, single nucleotide variants can alter functional levels, where amino acid changes can improve or repress the performance of proteins and, consequently, their biological functions in organisms with specific molecular signatures. Here, triploid Pacific oysters represent an excellent model for exploring whether triploidy induction alters the signatures of amino acid changes associated with single nucleotide variants and the biological functions where the mutations were identified throughout the genome. In parallel, SNPs have been observed to have potential effects on the spliceosome. Specifically, single mutations can disrupt the splice site, affecting the isoforms of mRNAs and likely the proteome in triploid oysters. This study aimed to explore the differences in the signatures of amino acid changes and the splice site effects of the differential SNP repertoire between diploid and triploid Pacific oysters. Whole genome resequencing was performed in two Pacific oyster families induced to triploidy. Eight diploid and eight triploid individuals were sampled for Illumina sequencing with 30x coverage for each family. A bioinformatics pipeline was designed for SNP calling, filtering by ploidy, and local realignment to improve the identification of nucleotide variations. The amino acid changes and splice site effects were analyzed using the latest genome version for the Pacific oyster, xbMagGiga1.1. Gene ontology analyses were performed on genes differentially annotated with SNP variants and putative splice effects. The results showed no difference in nucleotide mutations and heterozygous/homozygous proportions between 2n/3n oysters. However, nucleotide variations were identified in genes with different functions, where the splice site effects showed strong differences associated with ploidy in oysters. Notably, the assessment between oyster families reveals molecular signatures related to the genetic background, suggesting family-specific nucleotide variations. Future investigations may elucidate how single nucleotide variations drive the phenotype in 3n oysters exposed to environmental stress.