The rhesus macaque (Macaca mulatta) is a key species for advancing biomedical research. Like all draft mammalian genomes, the draft rhesus assembly (rheMac2) has gaps, sequencing errors and misassemblies that have prevented automated annotation pipelines from functioning correctly. Another rhesus macaque assembly, CR_1.0, is also available but is substantially more fragmented than rheMac2 with smaller contigs and scaffolds. Annotations for these two assemblies are limited in completeness and accuracy. High quality assembly and annotation files are required for a wide range of studies including expression, genetic and evolutionary analyses.We report a new de novo assembly of the rhesus macaque genome (MacaM) that incorporates both the original Sanger sequences used to assemble rheMac2 and new Illumina sequences from the same animal. MacaM has a weighted average (N50) contig size of 64 kilobases, more than twice the size of the rheMac2 assembly and almost five times the size of the CR_1.0 assembly. The MacaM chromosome assembly incorporates information from previously unutilized mapping data and preliminary annotation of scaffolds. Independent assessment of the assemblies using Ion Torrent read alignments indicates that MacaM is more complete and accurate than rheMac2 and CR_1.0. We assembled messenger RNA sequences from several rhesus tissues into transcripts which allowed us to identify a total of 11,712 complete proteins representing 9,524 distinct genes. Using a combination of our assembled rhesus macaque transcripts and human transcripts, we annotated 18,757 transcripts and 16,050 genes with complete coding sequences in the MacaM assembly. Further, we demonstrate that the new annotations provide greatly improved accuracy as compared to the current annotations of rheMac2. Finally, we show that the MacaM genome provides an accurate resource for alignment of reads produced by RNA sequence expression studies.The MacaM assembly and annotation files provide a substantially more complete and accurate representation of the rhesus macaque genome than rheMac2 or CR_1.0 and will serve as an important resource for investigators conducting next-generation sequencing studies with nonhuman primates.This article was reviewed by Dr. Lutz Walter, Dr. Soojin Yi and Dr. Kateryna Makova.