Background and objectives: Ten genes previously showing different evidence of association with multiple sclerosis have been selected to validate. Methods: Eleven polymorphisms were genotyped with the iPLEX™ Sequenom in a well-powered collection of Spanish origin including 2863 multiple sclerosis cases and 2930 controls. Results: Replication extended to the following polymorphisms: PKN2 (rs305217), GTF2B (rs7538427), EPHA4 (rs1517440), YTHDF3 (rs12115114), ANKFN1 (rs17758761) and PTPRM (rs4798571), which did not reach the threshold of significance in a follow-up of the first genome-wide association study (GWAS) conducted in multiple sclerosis; TMEM39A (rs1132200), which appeared as a newly identified susceptibility gene in the same study; a gene previously reaching GWAS significance in Italy, CBLB (rs9657904); IL12B (rs6887695, rs10045431), a susceptibility gene shared by diverse autoimmune diseases and, finally, another gene showing inconclusive association with multiple sclerosis, CNR1 (rs1049353). Conclusions: Pooled analysis corroborated the effect on MS predisposition of three genes: TMEM39A [rs1132200: p_M-H=0.001; OR_M-H (95% CI)= 0.84 (0.75–0.93)], IL12B [rs6887695: p_M-H=0.03; OR_M-H (95% CI)= 1.09 (1.01–1.17)] and CBLB [rs9657904: p_M-H=0.01; OR_M-H (95% CI)= 0.89 (0.81–0.97)].