Microsatellite primers were developed for the Pacific Northwest conifer, Chamaecyparis lawsoniana (A. Murray) Parl. (Cupressaceae), to enhance efficiencies in disease-resistance breeding and germplasm screening for gene conservation of this rare species. Using multiplexed massively parallel Illumina sequencing, we identified over 300,000 microsatellite-containing sequences from 2 million paired-end microreads. After stringent filtering and primer evaluation, we selected 11 primer pairs and used these to screen variation in 4 populations of C. lawsoniana. Loci show between three and ten repeats per locus, with an average of eight. Screening of these markers in the North American relative Callitropsis nootkatensis demonstrated limited marker transferability, but these markers could have utility in Asian species of Chamaecyparis. These genetic markers show high polymorphism and should provide a high level of individual discrimination for paternity analysis in defined pedigrees, and routine screening of wild variation in Chamaecyparis lawsoniana.