The second intron in the mitochondrial gene nad1 was surveyed using PCR, DNA sequencing, or Southern hybridization in 323 species (313 genera, 212 families) of seed plants. The intron was absent in all 22 species (22 genera, 8 families) of non-Pinaceae conifers studied, in Welwitschia mirabilis, and in seven angiosperms. Whereas absence of the intron in seven angiosperms and Welwitschiais likely due to seven independent losses when evaluated against the recently published multigene phylogenies, the lack of the intron in all non-Pinaceae conifers can be best explained by a single loss. These data suggest that the non-Pinaceae conifers represent a monophyletic group. We also conducted a phylogenetic analysis of seed plants using a combined data set of the partial exon and intron sequences of nad1 generated from this study and published sequences of mitochondrial cox1 and small subunit (SSU) rDNA, chloroplast rbcL, and nuclear 18S rDNA. The results supported the split of conifers into two groups: Pinaceae and non-Pinaceae conifers. The Gnetales were sister to Pinaceae, in agreement with the conclusion from other recent molecular phylogenetic studies that refute the anthophyte hypothesis.