A mosaic minisatellite region has been identified in the mitochondrial genome of Norway spruce (Picea abies). The array was composed of three tandem repeats PaTR1 (32 bp), PaTR2a (26 bp) and PaTR2b (26 bp). PaTR2a and PaTR2b differed by one base substitution. The analysis of 92 trees covering the whole natural distribution area of the species allowed detection of 11 length variants ranging from 131 bp to 447 bp. This high intra-specific polymorphism relies on variation in the number of the tandem repeats. Population genetic parameters estimated among 14 populations suggested high population differentiation (Gst = 0.749). The phylogenetic analysis of the 11 sequenced length variants has been performed using a parsimony approach. The topology of the tree showed a good association of groups with geographical origin and a low level of size homoplasy. The phylogenetic reconstruction also suggests that this minisatellite locus has mainly evolved by an increase in the repeat copy number.