Premise of the study: Recently released Pinus plastome sequences support characterization of 15 plastid Simple Sequence Repeat (ptSSR) loci originally published for P. contorta and P. thunbergii. This allows selection of loci for single-tube PCR multiplexed genotyping in any subsection of the genus. Methods: Unique placement of primers and primer conservation across the genus were investigated, and a set of six loci were selected for single-tube multiplexing. We compare interspecific variation between ptSSRs and nucleotide sequences of ycf1 then test intraspecific variation for ptSSRs using 911 samples in the P. ponderosa species complex. Results: The ptSSR loci contain mononucleotide and complex repeats with additional length variation in flanking regions. They are not located in hypervariable regions and most primers are conserved across the genus. A single PCR per sample multiplexed for six loci yielded 45 alleles in 911 samples. Discussion: The protocol allows efficient genotyping of many samples. The ptSSR loci are too variable for Pinus phylogenies but are useful for the study of genetic structure within and among populations. The multiplex method could easily be extended to other plant groups by choosing primers for ptSSR loci in a plastome alignment for the target group.