The Amazon sailfin catfish (Pterygoplichthys pardalis), which belongs to the Loricariidae family, is an invasive species that has caused massive damage to the ecological environment. However, a high-quality reference genome for this catfish species has not yet been reported. Here, we successfully assembled the first chromosome-level high-quality genome of P. pardalis using data produced from multiple sequencing platforms. The assembled genome contains 26 chromosomes, with a scaffold N50 of 49.47 Mb. Different evaluation methods indicated the high connectivity and accuracy genome we got. In total, 23 859 protein-coding genes were predicted in the genome, 22 169 (92.92%) of which were functionally annotated in public databases. Phylogenetic analysis showed that P. pardalis was clustered with all other catfish studied and diverged from their common ancestor 132.5 million years ago. Whole-genome collinearity analysis indicated the chromosome 6 of P. pardalis was aligned to two distinct chromosomes in Ameiurus melas, Pangasianodon hypophthalmus, and Ictalurus punctatus, suggesting the occurrence of potential chromosomal fusion/fission events. Furthermore, many immune system-related genes were expanded in the P. pardalis genome, which may have contributed to their adaptive traits to highly polluted environments and successful invasion. This study not only provides insights into the genetic basis of the successful invasion of P. pardalis, but also provides important data for comparative genomic analysis of P. pardalis in Siluriformes in the future.