The Alps are one of the most significant geographical barriers in Europe and several isolated Swiss and Italian valleys retain the distinctive Ladin and Romansch languages, alongside the modern majority of Italian and German languages. Linguistically, Ladin belongs to the Romance languages, but some studies on mitochondrial DNA (mtDNA) variation have suggested a major Middle Eastern component to their genealogical origin. Furthermore, an observed high degree of within-population diversity has been interpreted as reflecting long-standing differentiation from other European populations and the absence of a major bottleneck in Ladin population history. To explore these issues further, we examined Y chromosome and mtDNA variation in two samples of Ladin speakers, two samples of German speakers and one sample of metropolitan Italian speakers. Our results (1) indicate reduced diversity in the Ladin-speaking and isolated German-speaking populations when compared to a sample of metropolitan Italian speakers, (2) fail to identify haplotypes that are rare in other European populations that other researchers have identified, and (3) indicate different Middle Eastern components to Ladin ancestry in different localities. These new results, in combination with Bayesian estimation of demographic parameters of interest (population size, population growth rate, and Palaeolithic/Neolithic admixture proportions) and phylogeographic analysis, suggest that the Ladin groups under study are small genetically isolated populations (subject to strong genetic drift), having a predominantly European ancestry, and in one locality, may have a greater Palaeolithic component to that ancestry than their neighbours.