The development of molecular techniques as a taxonomic tool and their integration with information provided by other disciplines, has enhanced species discovery, facilitated species delimitation and produced invaluable data for inferring species phylogenies. Here, we provide an example of how DNA sequence data, together with morphometric, distributional and ecological information, assist in identifying and diagnosing previously overlooked lineages. The nocturnal, ground-dwelling spider genus Dysdera has colonized all the Macaronesian archipelagos, and has undergone a major diversification in the Canary Islands. A recent molecular phylogenetic analysis of Dysdera species from the eastern Canary Islands revealed deep genetic divergences among some populations, suggesting the existence of cryptic taxa. Here, we combine data from mitochondrial and nuclear loci with morphological and ecological evidence to delimit and formally describe three previously overlooked species: D. aneris sp. nov., endemic to the Salvage Islands; D. mahan sp. nov., distributed along coastal habitats of Lanzarote, north of Fuerteventura and adjacent islets; and D. simbeque sp. nov., restricted to two valleys in northern Lanzarote. Molecular markers provide key information that allows apparent morphological polymorphisms to be used as diagnostic features of evolutionarily independent lineages. Dysdera mahan sp. nov. is unique among the Canarian Dysdera in that it is found in the intertidal zone on pebbled beaches. Low levels of genetic variability and genital differentiation associated with relatively high somatic divergence suggest that speciation in D. mahan sp. nov. was driven by a selection of phenotypic traits that are adaptive to this rare environment. Separate analyses and statistical tests revealed phylogenetic incongruence between mitochondrial and nuclear genes, probably as a result of incomplete lineage sorting. The temporal framework for the origin and diversification of the new species inferred from the molecular data corroborates former hypotheses on the late Pliocene origin of the present-day biota of the Salvage Islands.