The evolutionary history of horseshoe crabs, spanning approximately 500 million years, is characterized by remarkable morphological stasis and a low species diversity with only four extant species. Here we report a chromosome-level genome assembly for the mangrove horseshoe crab (Carcinoscorpius rotundicauda) using PacBio reads and Hi-C data. The assembly spans 1.67 Gb with contig N50 of 7.8 Mb and 98% of the genome assigned to 16 chromosomes. The genome contains five Hox clusters with 34 Hox genes, the highest number reported in any invertebrate. Detailed analysis of the genome provides evidence that suggests three rounds of whole-genome duplication (WGD), raising questions about the relationship between WGD and species radiation. Several gene families, particularly those involved in innate immunity, have undergone extensive tandem duplication. These expanded gene families may be important components of the innate immune system of horseshoe crabs, whose amebocyte lysate is a sensitive agent for detecting endotoxin contamination.