Titin provides a molecular blueprint for muscle sarcomere assembly, and sarcomere length can vary according to titin isoform expression. If variations in sarcomere length influence muscle fascicle length, this may provide an advantage for running performance. Thus, the aim of this study was to investigate whether the titin (TTN) rs10497520 polymorphism was associated with muscle fascicle length in recreationally active men (RA; n=137) and marathon personal best time in male marathon runners (MR; n=141). Fascicle length of the vastus lateralis was assessed in vivo using B‐mode ultrasonography at 50% of muscle length in RA. All participants provided either a whole blood, saliva or buccal cell sample, from which DNA was isolated and genotyped using real‐time polymerase chain reaction. Vastus lateralis fascicle length was 10.4% longer in CC homozygotes, those carrying two copies of the C‐allele, than CT heterozygotes (P=.003) in RA. In the absence of any TT homozygotes, reflective of the low T‐allele frequency within Caucasian populations, it is unclear whether fascicle length for this group would have been smaller still. No differences in genotype frequency between the RA and MR groups were observed (P=.500), although within the MR group, the T‐allele carriers demonstrated marathon personal best times 2 minutes 25 seconds faster than CC homozygotes (P=.020). These results suggest that the T‐allele at rs10497520 in the TTN gene is associated with shorter skeletal muscle fascicle length and conveys an advantage for marathon running performance in habitually trained men.