© 2021, Società Italiana di Medicina Interna (SIMI).Peripheral lymphadenopathy (LAP) is an important and common abnormal finding of the physical exam in general medical practice. We aimed to reveal the LAP etiology and demographic, clinical and laboratory variables that may be useful in the differential evaluation of LAP. This multicenter, nested case–control study including 1401 patients between 2014 and 2019 was conducted in 19 tertiary teaching and research hospitals from different regions in Turkey. The ratio of infectious, malign and autoimmune/inflammatory diseases was 31.3%, 5% and 0.3%, respectively. In 870 (62%) of patients had nonspecific etiology. Extrapulmonary tuberculosis (n: 235, 16.8%) was the most frequent cause of LAP. The ratio of infective etiology of LAP was significantly lower in patients older than 65 years-old compared to younger patients with the rate of 66.67% and 83.84%, respectively (p 0.016, OR 0.386, 95% Cl 0.186–0.803). The probability of malign etiology was higher both in patients who are older than 45 years-old (p < 0.001, OR 3.23, 95% Cl 1.99–5.26) and older than 65 years-old (p 0.002, OR 3.36, 95% Cl 1.69–6.68). Age, localization and duration of LAP, leukocytosis, anemia, thrombocytopenia, CRP and sedimentation rate were important parameters to differentiate infections. Size of lymph node and splenomegaly in addition to the parameters above were useful parameters for differentiating malign from benign etiology. Despite the improvements in diagnostic tools, reaching a definite differential diagnosis of lymphadenopathy is still challenging. Our results may help clinicians to decide in which cases they need an aggressive workup and set strategies on optimizing the diagnostic approach of adulthood lymphadenopathy.