Skin prick tests (SPT) with latex extracts are commonly used in the diagnostic approach of natural rubber latex (NRL) allergy. Non-ammoniated latex extracts are commonly utilized for this purpose. There is only one commercially available ammoniated latex extract for both diagnostic and therapeutic purposes. To our knowledge, no direct clinical comparisons have been made so far comparing sensitivity and specificity of different latex extracts in patients with clinically confirmed latex allergy. The objective of this study was to evaluate sensitivity and specificity of non-ammoniated or ammoniated SPT of latex extracts in children with a clinical history suggestive of NRL allergy. Forty-two subjects (age 4–15 yr), referred to our Allergy Clinic Center, with suspected clinical signs of NRL allergy were evaluated. Patients were selected according to the following inclusion criteria: pediatric patients of age above 4 yr with a suspected clinical history of urticaria, rhinoconjunctivitis and/or asthma due to sensitization to NRL. A definitive NRL allergy diagnosis was made on the identification of latex-specific IgE (>0.7 kU/l), positive glove use test and a highly indicative clinical history. NRL allergy was confirmed in 26 (62%) subjects. SPT for latex were performed with three different SPT solutions containing non-ammoniated NRL extracts (product A: Lofarma SpA, Milan, Italy and product B: Stallergenes, Milan, Italy) or an ammoniated extract (product C: ALK-Abellò, Madrid, Spain). A positive result was considered as the appearance of a wheal with a diameter >3 mm. In patients with confirmed NRL allergy, wheal mean (s.d.) diameters were 7.3 (2.1) with product C, and 5.7 (2.3) and 4.4 (1.8) with products B and A respectively (p = 0.004 C vs. B and A). Mean wheal diameters were significantly higher for product C in comparison with product B and A (p < 0.04). Sensitivity according to the different products tested was 65% (product A); 81% (product B) and 96% (product C) (p = 0.0084; C vs. A). No difference between the three products was observed regarding diagnostic specificity (94%, 88% and 94%, product A, B and C, respectively). Our study shows that diagnostic sensitivity of different latex extract for SPT could differ. In this particular clinical setting (i.e., children with NRL allergy) ammoniated latex extract shows a higher sensitivity in comparison with non-ammoniated products. This aspect should be considered in the diagnostic approach in subjects with suspected NRL allergy. In children with clinical confirmed latex allergy, sensitivity and specificity of different commercially available skin prick test could vary. Ammoniated latex extract has shown a higher sensibility in comparison with non-ammoniated products.