MatK and rbcL are recommended as the official barcode loci for higher plants but there remains a need for additional universal markers. We generated a series of 84 new universal primers targeting 42 plastid loci that all yielded single amplicons when applied to DNA templates from 19 diverse higher plant families. Marker utility ultimately depends on sequence variability, with rapidly evolving loci being useful for barcoding or biogeographic applications and more conserved loci being better suited to deep phylogeny reconstruction. Whereas excessive size variation is undesirable for many applications, modest size variability caused by indels and the sequence variation frequently associated with indels are highly desirable. We therefore performed a quick screen of the markers for size and sequence variation using pooled DNA templates from 96 taxonomically diverse species. All markers produced little or no size variation (consistent with the presence of minor indels). The seven regions exhibiting most size variation in pooled templates (rpl23&rpl2.1, 16S, 23S, 4.5S&5S, petB&D and rpl2, rpoC1 and trnK introns) were then amplified for all species individually, confirming the pooled template results. When the most variable loci (introns of trnK and rpoC1) were sequenced for all 96 species, a high level of sequence variation (nucleotide substitutions and indels) was observed among congeneric species groups for both loci. Both markers therefore have potential as supplementary barcode markers.