Aleppo pine (Pinus halepensis Mill.) is an important component of Mediterranean forests. However, the scarcity of molecular resources limits population genetics studies shedding light on biogeographical patterns and/or within-population dynamics in this species. This is due to the complexity of conifers' genome, which includes many repetitive elements, and to the low genetic variation found in large parts of Aleppo pine distribution. The use of Next-Generation Sequencing (NGS) allows the development of molecular resources even for non-model species with complex genomes (i.e., complete genome and transcriptome sequencing, high numbers of neutral and putative functional markers, EST-SSRs, and SNPs). In this study, we developed a set of 14 polymorphic microsatellites (EST-SSRs) derived from transcriptome data, and combined them in three highly informative multiplexes for cost-effective genetic studies in P. halepensis. The three multiplexes were designed to allow addition of previously available SSRs of adequate size. Loci included in the EST-SSR multiplexes generally showed no null alleles and replicable, easily scorable amplification patterns. Furthermore, the cross-species transferability to two other Mediterranean pines (Pinus pinaster Aiton and Pinus heldreichii H. Christ 1863) was tested, revealing that these EST-SSRs are highly transferable and polymorphic across Mediterranean Pinus species. Finally, preliminary genetic analyses on multiple populations showed some potential applications of the newly developed EST-SSRs to population genetics studies within and among Mediterranean pine populations.