We describe a new Brookesia species from a forest fragment located 13 km south of Ambalavao in the southern part of Madagascar's central high plateau. Brookesia brunoi sp. nov. is one of the few arid-adapted Brookesia species inhabiting deciduous forests on the western slope of the central high plateau of the island (around 950 m a.s.l.). So far the species has only been observed in the private Anja Reserve. The species belongs to the Brookesia decaryi group formed by arid-adapt-ed Brookesia species of western Madagascar: B. bonsi Ramanantsoa, B. perarmata (Angel), B. brygooi Raxworthy & Nussbaum and B. decaryi Angel. Brookesia brunoi differs from the other four species of the group by a genetic divergence of more than 17.6% in the mitochondrial ND2 gene, and by a combination of morphological characters: (1) nine pairs of laterovertebral pointed tubercles, (2) absence of enlarged pointed tubercles around the vent, (3) presence of poorly defined laterovertebral tubercles along the entire tail, (4) by the configuration of its cephalic crest, and (5) hemipenial morphology. Based on our molecular phylogeny this species is sister to a clade containing B. brygooi, B. decaryi, and probably B. bonsi for which no ND2 sequences were available. Our molecular data also confirm the presence of a divergent mitochondrial lineage in the Tsingy de Bemaraha, which might be assigned to either B. bonsi or B. decaryi, and point to the need of more research on this population.