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Tsetse flies are obligate hematophagous vectors of animal and human African trypanosomosis. They cyclically transmit pathogenic <i>Trypanosoma</i> species. The endosymbiont <i>Sodalis glossinidius</i> is suggested to play a role in facilitating the susceptibility of tsetse flies to trypanosome infections. Therefore, this study was aimed at determining the prevalence of <i>S. glossinidius</i> and trypanosomes circulating in tsetse flies and checking whether an association exists between trypanosomes and <i>Sodalis</i> infections in tsetse flies from Kafue National Park in Zambia. A total of 326 tsetse flies were sampled from the Chunga and Ngoma areas of the national park. After DNA extraction was conducted, the presence of <i>S. glossinidius</i> and trypanosome DNA was checked using PCR. The Chi-square test was carried out to determine whether there was an association between the presence of <i>S. glossinidius</i> and trypanosome infections. Out of the total tsetse flies collected, the prevalence of <i>S. glossinidius</i> and trypanosomes was 21.8% and 19.3%, respectively. The prevalence of <i>S. glossinidius</i> was 22.2% in <i>Glossina morsitans</i> and 19.6% in <i>Glossina pallidipes</i>. In relation to sampling sites, the prevalence of <i>S. glossinidius</i> was 26.0% in Chunga and 21.0% in Ngoma. DNA of trypanosomes was detected in 18.9% of <i>G. morsitans</i> and 21.4% of <i>G. pallidipes</i>. The prevalence of trypanosomes was 21.7% and 6.0% for Ngoma and Chunga, respectively. The prevalences of trypanosome species detected in this study were 6.4%, 4.6%, 4.0%, 3.7%, 3.1%, and 2.5% for <i>T. vivax</i>, <i>T. simiae</i>, <i>T. congolense</i>, <i>T. godfreyi</i>, <i>T. simiae</i> Tsavo, and <i>T. b. brucei</i>, respectively. Out of 63 trypanosome infected tsetse flies, 47.6% of the flies also carried <i>S. glossinidius,</i> and the remaining flies were devoid of <i>S. glossinidius</i>. A statistically significant association was found between <i>S. glossinidius</i> and trypanosomes (<i>p</i> < 0.001) infections in tsetse flies. Our findings indicated that presence of <i>S. glossinidius</i> increases the susceptibility of tsetse flies to trypanosome infections and <i>S. glossinidius</i> could be a potential candidate for symbiont-mediated vector control in these tsetse species.