Background <p>Venom cocktails of cone snails are characterized by their dynamic composition and the rapid evolution of toxin-encoding genes due to various selective pressures. The integration of comparative genomics, transcriptomics, and proteomics has emerged as a crucial approach for the comprehensive characterization of venom repertoires and the understanding of venom gene evolution.</p> Results <p>The venom gland transcriptomes of three individuals of the Canary Island cone snail <i>Kalloconus canariensis</i> had 586 different transcripts, and showed remarkably high intraspecific variability. These transcripts were used to annotate and locate venom genes in the assembled pseudochromosomes of the <i>K. canariensis</i> genome, and comparative genomic analyses were performed with the venom genes of the closely related Mediterranean cone snail <i>Lautoconus ventricosus</i>. A total of 86 orthogroups with counterparts in both genomes were identified, comprising 118 and 159 venom genes from <i>K. canariensis</i> and <i>L. ventricosus</i>, respectively. Synteny analyses comparing the two genomes revealed that most (86%) venom genes were located in relatively conserved genomic regions within homologous pseudochromosomes. Notwithstanding, up to 16 genes were rearranged together with their flanking regions into non-homologous pseudochromosomes, suggesting local genomic reorganizations and/ or transpositions, likely mediated by transposable elements.</p> Conclusions <p>Comparative analyses of <i>K. canariensis</i> and <i>L. ventricosus</i> genomes revealed a core set of venom genes common to both species, showing overall synteny conservation. In addition, numerous instances of lineage-specific venom locus configurations were detected, suggesting that local tandem gene duplications and losses are major drivers of diversification for this gene family.</p>