Contiguous assemblies are fundamental to deciphering the composition of extant genomes. In molluscs, this is considerably challenging owing to the large size of their genomes, heterozygosity, and widespread repetitive content. Consequently, long-read sequencing technologies are fundamental for high contiguity and quality. The first genome assembly ofMargaritifera margaritifera(Linnaeus, 1758) (Mollusca: Bivalvia: Unionida), a culturally relevant, widespread, and highly threatened species of freshwater mussels, was recently generated. However, the resulting genome is highly fragmented since the assembly relied on short-read approaches. Here, an improved reference genome assembly was generated using a combination of PacBio CLR long reads and Illumina paired-end short reads. This genome assembly is 2.4 Gb long, organized into 1,700 scaffolds with a contig N50 length of 3.4 Mbp. Theab initiogene prediction resulted in 48,314 protein-coding genes. Our new assembly is a substantial improvement and an essential resource for studying this species’ unique biological and evolutionary features, helping promote its conservation.