Scalp pruritus is a common skin problem that remains therapeutic challenge. The relationships between the dysbiosis of microbiota and skin diseases have caught attention recently. However, there are few reports about microbiota on itchy scalp. This study investigated scalp microbial characteristics of subjects with mild scalp pruritus of undetermined origin and preliminarily screened physiological factors and bacteria potentially related to pruritus. The pruritus severity of 17 qualified females was evaluated by Visual Analogue Scale (VAS). Microbiota collection was done at both itchy (n = 20) and non-itchy sites (n = 27) at occiput and crown of the same subject and Illumina sequencing was performed at the V3–V4 hypervariable regions of 16S rRNA. The corresponding sebum content, hydration, pH, trans-epidermal water loss, erythema index and porphyrin numbers were also measured by skin tester. We identified 3044 amplicon sequence variants from 821 genera. The itchy and non-itchy sites had different microbiota structures (p = 0.045, by multivariate analysis of variance), while there were large inter- and intra-individual variations. Both sites had Staphylococcus, Cutibacterium and Lawsonella as predominant genera, which were not significantly related to pruritus. The use of three genera Lactobacillus, Morganella and Pseudomonas, could well distinguish non-itchy from itchy groups, whereas different composition patterns existed inside each group. Our investigation indicated that though the bacterial community structure on itchy scalp was individual specific, there was difference between itchy and non-itchy sites. The study provides new insights into microbiota profiling on itchy scalp, which will help microbiota-targeted therapeutic experiment or products design for scalp pruritus.