In 2016, we reported the presence of Plasmodium vivax in Botswana through active case detection. A real-time PCR was used during a similar study in 10 districts to assess changes in the P. vivax prevalence. We assessed 1,614 children (2-13 years of age) for hemoglobin (Hb; g/dL) and Plasmodium parasites. The median age of all participants was 5.0 years (25th percentile, 3 years; 75th percentile, 8 years). The median Hb (g/dL) level was 12.1, but 18.3% of the participants had anemia (Hb < 11.0 g/dL); these participants were clustered in the younger than 5 years age group in all districts (P < 0.001). The risk of anemia decreased with age 5 years or older (odds ratio [OR], 0.26; 95% confidence interval [CI], 0.197-0.34; P < 0.001). The prevalence rates of Plasmodium parasites were as follows: P. vivax, 12.7%; P. falciparum, 12.7%; P. malariae, 0.74%; and P. ovale (P. ovale curtisi), 0.68%. Mixed infection rates were as follows: P. falciparum and P. vivax, 2.35%; P. falciparum and P. ovale curtisi, 0.56%; P. vivax and P. malariae, 0.06%; and P. falciparum and P. malariae, 0.68%. The infections were largely asymptomatic (99.6%). Using logistic regression, the risk of infection with P. vivax was highest in Kweneng East (OR, 6.2; 95% CI, 2.9-13.1), followed by South East (OR, 5.6; 95% CI, 2.5-12.3) and Ngami (OR, 5.1; 95% CI, 2.2-12.0). Compared to the risk of infection for children younger than 5 years, the risk of infection decreased for children 5 years or older in regions with high rates of P. vivax and P. falciparum infections. P. vivax and P. falciparum have expanded within the asymptomatic population in Botswana; therefore, careful attention is required for their elimination.