Background <p>Eurasian red squirrels (<i>Sciurus vulgaris</i>) are common in Europe and Asia, but are declining in many regions across their range. As continental European populations are now facing current and future threats from invasive species in addition to existing anthropogenic pressures it will be important to carefully monitor these populations. Non-invasive genetic sampling methods are a useful tool in conservation assessments, but often require techniques such as microsatellite markers that can be used with lower quality DNA. It remains helpful to increase the resolution of these assessments by identifying additional genetic markers.&#xa0;We describe new microsatellite markers developed from European red squirrels from France and use them to assess genetic diversity in populations in southern France.</p> Methods and results <p>We used Illumina sequencing to characterize microsatellites from tissue samples of <i>S. vulgaris</i>. Using 7 tissue samples we assessed amplification and polymorphism in 48 microsatellite inserts and further evaluated 16 of these microsatellite loci in hair samples from 120 individuals from four populations. In the 104 samples for which those loci amplified, there was an average of 6.1 alleles amplified per locus, with mean observed and expected heterozygosities of 0.44 and 0.59, respectively. Only one locus showed significant deviation from HWE across all populations. The same locus exhibited a likely presence of null alleles.</p> Conclusions <p>We describe 16 new microsatellite loci, with caution required for one locus in analyses sensitive to null alleles. These new loci can help provide increased resolution in population genetic assessments of red squirrels in continental Europe.</p>