The two native Croatian donkey breeds (Littoral-Dinaric donkey and Istrian donkey) were marginalized in the second half of the 20th century and were on the verge of biological extinction. The aim of this study was to analyze the demographic and genetic status of two donkey breeds, two decades after the start of protection by analyzing their pedigrees and genetic structure. The average generation interval was higher for the Istrian donkey (7.73) than for the Littoral-Dinaric donkey (7.27). The rate of the effective number of founders compared with the effective number of ancestors in the Littoral-Dinaric donkey (1.03; 325/316) and in the Istrian donkey (1.08; 70/65) revealed no evidence of a genetic bottleneck. The inbreeding coefficient (F) and the average relatedness coefficient (AR) was lower in the Littoral-Dinaric donkey population (0.99%; 0.13%) than in the Istrian donkey population (1.77%; 1.10%). Genetic microsatellite analysis showed relatively high genetic diversity in Littoral-Dinaric donkey and Istrian donkey breeds, expressed by mean allele number (5.92; 5.85) and expected heterozygosity (0.650; 0.653). Genetic differentiation between the Littoral-Dinaric donkey and the Istrian donkey has not significantly increased in the last two decades (FST = 0.028). Genetic analysis also showed no evidence of high inbreeding or genetic bottleneck in both breeds. A total of 11 haplotypes including 28 polymorphic sites were found in 30 samples. Analysis of mtDNA has shown that the Littoral-Dinaric donkey and Istrian donkey breeds belong to the Equus asinus africanus group. The study confirms the need to use different analytical approaches to get a regular and complete insight into the situation and trends within and between breeds, so that the existing diversity can be fully preserved.