Prior work in the boreal forest unearthed a novel ITS-LSU sequence (NS1) that falls outside known fungal phyla. Here we performed a targeted PCR survey to investigate the ecology of NS1. NS1 was found in three out of 99 soil cores at one black spruce (Picea mariana) site, and two cores were from nearby subplots, but clumping could not be demonstrated statistically. However, NS1 was detected 6 yr later in the same subplot, and in an adjacent subplot, with a join count probability of 0.0073. NS1 was not found in other lowland black spruce sites, but was detected in several upland mixed hardwood/white spruce (Picea glauca) sites and was correlated with presence of white spruce (p = 0.0011). It was also found in the same upland sites sampled in consecutive years. Our results provide clues concerning the ecology of NS1 and suggest that rare, divergent sequences should not necessarily be discarded from environmental sequence datasets.