A collection of 141 old and local Spanish accessions of pear (Pyrus communis) from the Escuela Tecnica Superior de Ingenieria Agraria-Universidad de Lleida (ETSIA-UdL) Pear Germplasm Bank in Lleida, Spain, were studied using a set of eight microsatellite markers to estimate the genetic diversity of the collection, to identify the genetic structure and relationships among its accessions, and to establish a representative core collection. An additional set of 13 well-known pear cultivars, currently grown in Spain and which represent a wide genetic diversity, were added as reference. The eight simple sequence repeat (SSR) loci amplified 97 alleles, with nine to 15 alleles per locus, and with the expected heterozygosity ranging from 0.65 to 0.89. All of the accessions except for 16 had at least one of the 48 rare alleles (frequency < 0.05) identified, and seven unique alleles were found in six accessions. Fifteen accessions were identified as synonyms and were excluded from the analysis. Genetic analyses performed by hierarchical clustering, Bayesian model-based clustering, and factorial correspondence analysis supported the existence of three groups among the accessions with moderate [fixation index (F-ST) = 0.074], but significant, differentiation. As a whole, most of the germplasm (about 75%) curated at the collection showed its genetic distinctness with respect to the main pear cultivars used in European orchards. In fact, most reference cultivars were included in one single cluster that, moreover, had the lowest genetic diversity and allelic richness, in spite of having been chosen as heterogeneous material from different origins. The obtained results were also used to create a core collection with 35 accessions constituting an efficient and accessible entry point in the ETSIA-UdL pear collection for breeding and research communities.