Serotonin is a multifunctional signaling molecule involved in a wide range of physiological functions, with a particularly prominent role in regulating the body's energy balance. Serotonin signaling is largely modulated by the serotonin transporter, a transmembrane protein through which serotonin enters the cell, what leads to termination of the serotonin signaling. Certain polymorphisms of the SLC6A4 gene, coding for the serotonin transporter, are associated with the intensity of transcription of this gene and with its susceptibility to the development of obesity and related comorbidities. The main goal of this study was to investigate the potential association of SLC6A4 gene polymorphisms with neonatal anthropometric characteristics, taking into account the possible influence of different neonatal and maternal characteristics. Genomic DNA was isolated from placental tissue and genotyping of the 5-HTTLPR promoter polymorphism and STin2 intron polymorphism was performed by polymerase chain reaction (PCR), while genotyping of the single nucleotide polymorphism rs25531 was performed by digestion of the obtained 5-HTTLPR PCR products with restriction endonuclease MspI. A method optimisation procedure was carried out as part of the study, which included modification of the PCR and agarose gel electrophoresis methods. The determined frequencies of the genotypes and alleles of the analyzed polymorphisms were in accordance with the previously observed in the Croatian population. A statistically significant effect of sex and gestational age of the newborns on birth weight and length, but not on the ponderal index, was observed. A statistically significant difference in the ponderal index was found between newborns of different genotypes for the STin2 polymorphism, both in the total sample and in the group of female, but not male newborns. Understanding the underlying influence of biological and other factors on the neonatal anthropometric characteristics could contribute to the development of programs of early intervention and prevention of metabolic disorders and accompanying comorbidities later in life.