Evolutionary reconstruction of the natural history of an organism ultimately requires knowledge about the development, population genetics, ecology, and phylogeny of the species. Such investigations would benefit from studies of mutational processes because mutations are the source of natural variation. The nematode Pristionchus pacificus has been developed as a model organism in evolutionary biology by comparing its development with Caenorhabditis elegans. Pristionchus pacificus and related species are associated with scarab beetles, and their ecology and phylogeny are well known. More than 200 P. pacificus isolates from all over the world are available for this cosmopolitan species. We generated mutation accumulation (MA) lines in P. pacificus to study spontaneous mutation rates in the mitochondrial genome and compared mutation rate estimates with natural variation between nine representative isolates of the species. The P. pacificus mitochondrial genome is 15,955 bp in length and is typical for nematodes. Pristionchus pacificus has all known mitochondrial genes and contains an unusual suppressor transfer RNA (tRNA) for the codon UAA. This has most likely influenced the spectrum of observable mutations because 6 of 12 mutations found in the 82 MA lines analyzed are nonsense mutations that can be suppressed by the suppressor tRNA. The overall mutation rate in P. pacificus is 7.6 × 10⁻⁸ per site per generation and is less than one order of magnitude different from estimates in C. elegans and Drosophila. Using this mutation rate estimate in a comparison of the mitochondrial genome of nine P. pacificus isolates, we calculate the minimum time to the most recent common ancestor at 10⁵-10⁶ generations. The combination of mutation rate analysis with intraspecific divergence provides a powerful tool for the reconstruction of the natural history of P. pacificus, and we discuss the ecological implication of these findings.