The cophyline microhylid frog genus Anilany was established as a monotypic genus in 2016 for the miniaturised species Anilany helenae (Vallan, 2000), from the type locality Ambohitantely, a patch of rainforest surrounded by savannah in central Madagascar. Fieldwork conducted over the past two decades identified three unexpected populations from Bemaraha, Mahajanga, and Beanka from limestone caves near sea level in arid western and northwestern Madagascar, which were assigned to the genus Anilany based on diagnostic morphological features. We generated new data for specimens of the Ambohitantely, Bemaraha, and Mahajanga populations to test if all three populations belong to one or multiple species, studying their genetic variability based on mitochondrial (16S rRNA, COI) and nuclear (BDNF, RAG-1) genes, as well as morphological and osteological data. We identify several previously unknown diagnostic characters of the skeleton of Anilany compared to its closest relatives, Rhombophryne and Stumpffia, but no skeletal features that can be used to reliably distinguish among Anilany populations. Based on concordant genetic and morphological differences, we formally describe the population from the limestone karsts of Tsingy de Bemaraha National Park, formerly considered candidate species Anilany sp. Ca14, as a new species, Anilany karsticola sp. n. It can be distinguished from other lineages by larger size and shorter relative tibia length, uncorrected p-distances in DNA sequences of the 16S gene amounting to 2.6% and a lack of allele sharing in the analysed fragments of the nuclear genes BDNF and RAG-1. More data, especially advertisement call recordings and additional specimens, are required from other locations for a more thorough assessment of the genus and the distribution of its species.