Treacher Collins syndrome (TCS) is a rare congenital craniofacial disorder with variable penetrance and high genetic and phenotypic heterogeneity. It is caused by pathogenic variants in the TCOF1, POLR1D, POLR1C, and POLR1B genes, and its major characteristic features are malar and mandibular hypoplasia, downward slanting of the palpebral fissures, and conductive hearing loss. In this study, five patients (two males and three females, age range from 2 to 29 years) with TCS were tested by Next-Generation Sequencing (NGS)-based sequencing and clinically characterized. Genetic analyses detected two deletions and one insertion in the TCOF1 gene and one missense variant in the POLR1D gene. Two novel mutations, c.1371_1372insT (p.Lys458*) in the TCOF1 gene and c.295 G>C (p.Gly99Arg) in the POLR1D gene, were identified. Moreover, two already known mutations, c.4369_4373del (p.Lys1457Glufs*12) and c.2103_2106del (p.Ser701Argfs*9) in the TCOF1 gene, were detected. The novel TCOF1 c.1371_1372insT mutation was associated with mild craniofacial manifestations and very rare symptoms of TCS, i.e., developmental delay and moderate intellectual disability. Although incomplete penetrance is a known phenomenon in TCS, surprisingly, the majority of our patients inherited the disease-causing variants from an asymptomatic mother. The unique feature of our study is the observation of causative mutation transmission between asymptomatic family members. Our results expanded the clinical and mutational spectrum of TCS and further confirmed the inter- and intra-familial variability of this disorder.