Wolbachia are vertically transmitted endosymbiotic bacteria of arthropods and onchocercid nematodes. It is commonly accepted that they co-evolved with their filarial hosts, and have secondarily been lost in some species. However, most of the data on the Wolbachia/Onchocercidae relationship have been derived from studies on two subfamilies, the Dirofilariinae and the Onchocercinae, which harbour parasites of humans and domestic animals. Within the last few years, analyses of more diverse material have suggested that some groups of Onchocercidae do not have Wolbachia, such as recently studied Splendidofilariinae from birds. This study takes advantage of the analysis of additional Splendidofilariinae, Rumenfilaria andersoni from a Finnish reindeer and Madathamugadia hiepei from a South African gecko, using PCR, immunohistochemical staining and whole-mount fluorescent analysis to detect Wolbachia and describe its strains. A DNA barcoding approach and phylogenetic analyses were used to investigate the symbiosis between Wolbachia and the Onchocercidae. A new supergroup F Wolbachia was demonstrated in M. hiepei, representing the first filarial nematode harbouring Wolbachia described in a non-mammalian host. In the adult, Wolbachia infects the female germline but not the hypodermis, and intestinal cells are also infected. The phylogenetic analyses confirmed a recent emergence of supergroup F. They also suggested several events of horizontal transmission between nematodes and arthropods in this supergroup, and the existence of different metabolic interactions between the filarial nematodes and their symbionts.