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 504 wild trees and 417 breeding individuals from the Dorena Genetic Resource Center (DGRC). Wild populations of Port-Orford-cedar (POC) show a high degree of genetic differentiation across the landscape, with 25% of overall variation attributable to sampling locations. Bayesian clustering methods reveal that genetic variation is best modeled by eight groups, and these groups show strong geographic clustering, particularly in the southern end of the species range. Our analysis of DGRC seed orchard populations shows that six of the eight Bayesian classes are well represented in the collection. Two Bayesian classes that originate primarily from the southern end of the range (California north coast; California Klamath range) are underrepresented in the DGRC collection and should be targeted for future seed collection and gene conservation activities. These conifer microsatellite sequences can be accessed at OpenWetWare (http://openwetware.org/wiki/conifermicrosat).