Hexanucleotide repeats expansion in the C9orf72 gene is the most common cause of familial and sporadic amyotrophic lateral sclerosis (ALS) cases in Europe. In this study we aimed to determine the size and distribution of C9orf72 alleles, and investigate the possible association of the repeat size with several clinical parameters in ALS patients from Serbia. Patients were recruited from 2011-2021 and analysed using fragment length analysis and Southern blot. Out of 383 ALS patients, we have detected 31 (8.09%) patients with repeat expansion. In the total ALS cohort, clinical overlap with frontotemporal dementia (FTD) was registered in 17 (4.44%) patients, and among them, 5 (29.41%) were expansion carriers. There was no difference in the age of onset, age at the examination or disease duration, gender, and the frequency of spinal and bulbar onset between patients with and without C9orf72 expansion. The presence of positive family history (34.48% vs. 15.65%) and FTD (16.13% vs. 3.41%) was more frequent in expansion-positive vs. expansion-negative patients. In expansion-positive patients, significantly higher values of the largest detected repeat were found in patients with ALS in the family, and in expansion-negative, a higher median value of the smaller allele was noted in patients with a positive family history of ALS, dementia, and both in comparison to the rest of the group. A correlation of the repeat size was not found with the age of onset in both patients with and without the expansion. This is the first detailed study of C9orf72 sizing in ALS patients from Serbia. Our results emphasize the need for C9orf72 genetic screening in ALS patients with/without FTD.