We examined the amount and nature of variation revealed by cDNA-based sequence-tagged-site (STS) markers in Norway spruce (Picea abies (L.) Karst.) using 39 pairs of heterologous primers that were based upon arbitrary genes in black spruce (Picea mariana (Mill.) B.S.P.). A panel of 22 diverse Norway spruce genotypes was screened for variation that could be observed directly using standard agarose gel electrophoresis, without additional manipulation of amplification products. Examination of marker segregation among haploid megagametophytes revealed that nine markers behaved in a codominant manner, two markers had codominant length polymorphisms and null alleles, and four others had dominant length polymorphisms. DNA sequencing of codominant alleles at seven loci indicated that most insertions/deletions (indels) were in noncoding regions and that alleles often differed by the presence or absence of direct repeats that ranged in size from three to 23 bp. The nine markers that showed exclusively codominant polymorphisms in Norway spruce had an average observed heterozygosity of 0.30 and an average of 2.9 alleles in the panel of 22 trees. These levels of variation are similar to those previously found for similar sets of markers in other spruces, and appear to be at least as high as those revealed by polymorphic allozyme markers in Norway spruce. Polymorphisms at one STS locus suggested a higher affinity between Norway spruce and white spruce (Picea glauca (Moench) Voss) than between either of these spruces and black spruce. The STS markers described in this report should be useful in a variety of applications in Norway spruce, including population studies and genome mapping.