Abstract Catfish represents a diverse lineage with variable number of chromosomes and complex relationships with humans. Although certain species pose significant invasive threats to native fish populations, comprehensive genomic investigations into the evolutionary adaptations that contribute to their invasion success are lacking. To address this gap, our study presents a high-quality genome assembly of the Amazon sailfin catfish (Pterygoplichthys pardalis), a member of the armored catfish family, along with a comprehensive comparative genomic analysis. By utilizing conserved genomic regions across different catfish species, we reconstructed the 29 ancestral chromosomes of catfish, including two microchromosomes (28 and 29) that show different fusion and breakage patterns across species. Our analysis shows that the Amazon sailfin catfish genome is notably larger (1.58 Gb) with more than 40,000 coding genes. The genome expansion was linked to early repetitive sequence expansions and recent gene duplications. Several expanded genes are associated with immune functions, including antigen recognition domains like the Ig-v-set domain and the tandem expansion of the CD300 gene family. We also identified specific insertions in CNEs (conserved non-coding elements) near genes involved in cellular processes and neural development. Additionally, rapidly evolving and positively selected genes in the Amazon sailfin catfish genome were found to be associated with collagen formation. Moreover, we identified multiple positively selected codons in hoxb9, which may lead to functional alterations. These findings provide insights into molecular adaptations in an invasive catfish that may underlie its widespread invasion success.