Thymic stromal lymphopoietin (TSLP), an epithelium-derived pro-inflammatory cytokine, activates distinct immune and non-immune cells. It has been shown to be a master regulator of type 2 immune responses. Limited information is available on TSLP in childhood asthma. The aim of the present study was to find out whether there is association between TSLP concentrations and asthma phenotypes or disease activity. A total of 207 children with asthma and 100 healthy children aged 1–13 years were enrolled. This study examined serum TSLP concentrations using ELISA Kit in asthma patients and controls, analyzed its correlation with asthma phenotypes and pulmonary function. We also examined TSLP concentrations in 23 patients during stable asthma and in acute asthma exacerbation. The serum concentrations of TSLP were significantly elevated in asthma patients compared with healthy controls (p < 0.05), but there was no significant difference (p > 0.05) in TSLP concentrations between three different asthma phenotypes (allergic asthma, virus induced asthma and nonallergic asthma). There was no significant correlation between TSLP concentrations and FEV1pred% (r = 0.01, p > 0.05). In the acute asthma exacerbation TSLP concentrations were not significantly different than in stable phase of disease (p > 0.05). Children with asthma have higher serum TSLP concentrations when compared to healthy controls. TSLP does not seem to be a biomarker of disease exacerbation in children. Different asthma phenotypes have similar TSLP concentration profile in peripheral blood and TSLP does not seem to be useful biomarker in asthma phenotyping in children. •The thymic stromal lymphopoietin is a master regulator of type 2 immune responses.•Serum TSLP concentrations are higher in children with asthma.•TSLP role in identifying children with different asthma phenotypes is limited.•Serum TSLP does not seem to be a biomarker of asthma exacerbation.