The Attachment Insecurity Screening Inventory (AISI) 2-5 years is a parent-report questionnaire for assessing attachment insecurity in preschoolers. Validity and reliability of the AISI 2-5 years were examined in a general sample (n = 429) and in a clinical sample (n = 71). Confirmatory factor analysis (CFA) confirmed a three-factor model of avoidant, ambivalent/resistant and disorganized attachment, and one higher-order factor of total attachment insecurity. Multi-group CFA indicated measurement invariance across mothers and fathers, and across the general and clinical population sample. Reliability coefficients were generally found to be good. We found partial support for convergent validity in associations between AISI-scores and observed attachment (AQS). Concurrent validity was supported by associations between AISI-scores and observed parental sensitivity (MBQS) and parent-reported psychopathology (SDQ). Finally, the AISI discriminated well between children from the general and from the clinical sample. We argue that both research and practice could benefit from the AISI as there is now a prospect of quickly, reliably and validly screening for attachment insecurity in pre-school aged children. Based on this information, help can be offered timely and, subsequently, the prevention of attachment related problems of children can be strengthened.