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<i>Toxoplasma gondii</i> is a protozoan parasite responsible for human toxoplasmosis. The three major clonal lineages and different recombinant strains of <i>T. gondii</i> have a varied global distribution. This study aimed at evaluating the epidemiological distribution of types II and I–III and recombinant or mixed <i>T. gondii</i> in Italians and foreigners residing in Italy, establishing an association between serotypes and demographic characteristics. We collected the sera of 188 subjects who had tested positive for specific <i>T. gondii</i> antibodies. The population was differentiated into groups based on sex, nationality, and place of birth (Italy, Africa, South America, Asia, or Europe (except Italy)). We then performed a homemade ELISA test that detected both the antibodies against the amino acid sequences of the three main genotype antigens (I–III) in human sera and discerned the <i>T. gondii</i> strains. Serotype II of <i>T. gondii</i> was the most prevalent in the Italian population, whereas type I–III was the most prevalent in the foreign group. Surprisingly, we observed a notable amount of recombinant or mixed serotypes in European and Italian subjects. Moreover, we showed a significant difference in the prevalence of <i>T. gondii</i> serotypes between men and women, Italians, and foreigners. This descriptive study is the first to investigate the epidemiological distribution of <i>T. gondii</i> serotypes in humans in Italy using a homemade ELISA. We considered this technique suitable for discriminating between serotypes II and I–III and, consequently, for an epidemiological study focusing on the observation of circulating <i>T. gondii</i> strains and clinical correlations.