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Abstract Chlamydia trachomatis (CT) may employ persistence to evade antimicrobial clearance, possibly residing in the gastrointestinal tract. This study assessed the reliability of droplet digital PCR (ddPCR) in CT detection, its functionality in viability assessment, and predictions on CT transmission dynamics by combining viability PCR (vPCR) and clinical data from 52 infected women. The ddPCR showed 94% positive and 100% negative agreement with Abbott Alinity STI-M for endocervical samples, and 92% positive and 87% negative agreement in rectal samples. Viability was higher in endocervical samples (89.1%) than in rectal samples (69.4%). Samples from participants not engaging in anal intercourse, and with non-concordant multi-locus sequence typing between rectum and endocervix, had on average the highest viability in rectum, indicating a persistent population residing in the gastrointestinal tract. This study demonstrates the effectiveness of ddPCR in detecting CT, especially in samples with high inhibition or low bacterial load, suggesting its superiority over quantitative real-time PCR. These findings support that rectal CT infection can occur independently of anal intercourse, possibly through vaginorectal contamination or oral routes. High rectal CT viability, independent of endocervical infection, indicates potential gastrointestinal establishment. Understanding CT dynamics in various infection sites can provide insights into the epidemiology and pathogenesis of CT.