Background: The iris as a unique identifier is predicated on the assumption that the iris image does not alter. This does not consider the fact that the iris changes in response to certain external factors including medication, disease, surgery as well as longer term ageing changes. It is also part of a dynamic optical system that alters with light level and focussing distance. A means of distinguishing the features that do not alter over time from those that do is needed. This paper applies iris recognition algorithms to a newly acquired database of 186 iris images from four subjects. These images have greater magnification and detail than iris images in existing databases. Iris segmentation methods are tested on the database. A new technique that enhances segmentation is presented and compared to two existing methods. These are also applied to test the effects of pupil dilation in the identification process. Findings:Segmentation results from all the images showed that using the proposed algorithm accurately detected pupil boundaries for 96.2% respectively of the images, which was an increase of 88.7% over the most commonly used algorithm. For the images collected, the proposed technique also showed significant improvement in detection of the limbal boundary compared to the detection rates using existing methods. With regard to boundary displacement errors, only slight errors were found with the proposed technique compared to extreme errors made when existing techniques were applied. As the pupil becomes more dilated, the success of identification is increasingly more dependent on the decision criterion used. Conclusions:The enhanced segmentation technique described in this paper performs with greater accuracy than existing methods for the higher quality images collected in this study. Implementation of the proposed segmentation enhancement significantly improves pupil boundary detection and therefore overall iris segmentation. Pupil dilation is an important aspect of iris identification; with increasing dilation, there is a greater risk of identification failure. Choice of decision criterion for identification should be carefully reviewed. It needs to be recognised that differences in the quality of images in different databases may result in variations in the performance of iris recognition algorithms.