BACKGROUND: Functional characterisation of the compact genome of the model organism Caenorhabditis elegans remains incomplete despite its sequencing 20 years ago. The last decade of research has seen a tremendous increase in the number of non-coding RNAs identified in various organisms. While we have mechanistic understandings of small non-coding RNA pathways, long non-coding RNAs represent a diverse class of active transcripts whose function remains less well characterised. RESULTS: By analysing hundreds of published transcriptome datasets, we annotated 3392 potential lncRNAs including 143 multi-exonic loci that showed increased nucleotide conservation and GC content relative to other non-coding regions. Using CRISPR/Cas9 genome editing, we generated deletion mutants for ten long non-coding RNA loci. Using automated microscopy for in-depth phenotyping, we show that six of the long non-coding RNA loci are required for normal development and fertility. Using RNA interference-mediated gene knock-down, we provide evidence that for two of the long non-coding RNA loci, the observed phenotypes are dependent on the corresponding RNA transcripts. CONCLUSIONS: Our results highlight that a large section of the non-coding regions of the C. elegans genome remains unexplored. Based on our in vivo analysis of a selection of high-confidence lncRNA loci, we expect that a significant proportion of these high-confidence regions is likely to have a biological function at either the genomic or the transcript level.