Active optical sensors (AOSs) are used for in-season variable-rate application of nitrogen (N). The sensors measure crop reflectance expressed as vegetative indices (VIs). These are transformed into N recommendations during on-site calibration of AOSs—‘familiarising’ the sensors with the crop N status of the representative part of a field. The ‘drive-first’ method is often used by growers to calibrate AOSs. Due to large spatial variation of crop N status within fields, it is difficult to identify the most representative sample strip for AOS calibration. Seven site-years were used to evaluate the sensitivity of sensor-based N prescriptions for winter wheat (Triticum aestivum L.) to selection of sample strips for AOS calibration that fall into extreme, very low or very high values of 95th percentiles of amber normalised difference VI (NDVI) values. A Crop Circle ACS-210 sensor was used to collect canopy reflectance values, expressed as amber NDVI, at the beginning of wheat stem elongation. Our study showed that the sample-strip selection significantly affected sensor-based N prescriptions. The drive-first method may result in under- or over-applications of N and in lower N-use efficiency. One way to overcome this problem is to collect whole field NDVI values during pesticide application before sensor-based N application. The NDVI values from the entire field then can be used to choose the most representative sample strips for AOS calibration.